EGFR x CD28 MULTISPECIFIC ANTIBODIES

ABSTRACT

The present invention provides multispecific antibodies that bind to EGFR and CD28 (EGFRxCD28) as well as anti-EGFR antibodies. Such antibodies may be combined with a further therapeutic agent such as an anti-PD1 antibody. Methods for treating cancers (e.g., EGFR-expressing cancer) by administering the antibodies (e.g., and combinations thereof with anti-PD1) are also provided. The EGFRxCD28 antibodies of the present invention embody a tumor-targeted immunotherapeutic modality combined with PD-1 inhibition. These bispecific antibodies bind a tumor-specific antigen (TSA) (EGFR) with one arm and the co-stimulatory receptor, CD28, on T-cells with the other arm. Combination therapy with PD-1 inhibitors specifically potentiated intra-tumoral T cell activation, promoting an effector memory-like T cell phenotype without systemic cytokine secretion in a variety of syngeneic and human tumor xenograft models. Combining this class of CD28-co-stimulatory bispecific antibodies with the clinically validated anti-PD-1 treatment provides a well-tolerated antibody therapy with markedly enhanced anti-tumor efficacy.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/825,179, filed on Mar. 20, 2020, which is related to and claimspriority of U.S. Provisional Application No. 62/822,124, filed on Mar.22, 2019. The entire contents of each of the foregoing applications,including drawings and sequence listings, are expressly incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind to EGF receptorand CD28 and methods of use thereof, e.g., for treating or preventingcancer.

SEQUENCE LISTING

The instant application contains a Sequence Listing XML which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Feb. 6, 2023, isnamed 118003-10603.xml and is 105,147 bytes in size.

BACKGROUND OF THE INVENTION

The ability of T-cells to recognize and kill their cellular targets—suchas virally-infected cells or tumor cells—depends on a coordinated set ofinteractions. Foremost among these is the recognition and binding of thetarget cell by the T-cell Receptor (TCR) complex (which includes theassociated CD3 γ, δ, ε and ξ (chains), and this interaction has beenreferred to as “signal 1” for T-cell activation. The TCR recognizes aviral or tumor peptide presented on the groove of an MHC proteinexpressed on the surface of the target cell. Because such binding isgenerally of low-affinity, successful triggering of “signal 1” requiresclustering of many TCR complexes along the interface between the T-celland its target cell; this interface has been referred to as the “immunesynapse”. T-cell activation can be further promoted by additionalinteractions. For example, T-cells have a molecule referred to as CD28on their surface, which can provide a co-stimulatory “signal 2” toaugment the activation via the TCR complex. When a T-cell recognizes itstarget cell via its TCR complex, and then also engages “signal 2” viaCD28 binding to its cognate ligand(s) on the target cell, T-cellactivation is enhanced; as with “signal 1”, CD28-mediated “signal 2” isthought to occur via co-clustering at the immune synapse.

Agonistic anti-CD28 mAbs can be applied in sustained ex vivo expansionof cultured T-cells; however, the use of antibodies against CD28 hasbeen discouraged as a result of a series of acute and serious adverseevents in a phase I clinical trial where super agonist anti-CD28 mAb wastested systemically (Hunig, Nature Reviews Immunology. 2012;12:317-318). Localized or targeted use of anti-CD28 mAb can be used withless risk for promotion of antitumor immunity. Jung et aL., Int JCancer. 2001 Jan. 15; 91(2):225-30.

Different families of growth factors and growth factor receptors havebeen shown to be involved in the autonomous growth of cancer cells.Among these, the epidermal growth factor receptor (EGFR) and theEGF-family of peptide growth factor have a central role in thepathogenesis and progression of different carcinoma types. EGFR belongsto a family of receptors that encompasses three additional proteins,ErbB-2, ErbB-3 and ErbB-4. These proteins and the growth factors of theEGF family form an integrated system in which a signal that hits anindividual receptor type is often transmitted to other receptors of thesame family.

Monoclonal antibodies (mAbs) aimed at enhancing T-cell activation areunder clinical development as anti-tumor therapeutics. The majority ofcurrent treatments, however, have a difficult time overcoming theinhibitory nature of the tumor microenvironment, thus failing togenerate efficient tumor-specific T-cell activation and subsequent tumorcell killing. Several blocking mAbs directed against checkpointinhibitors such as CTLA-4 (cytotoxic T lymphocyte-associated protein)and programmed cell death 1 (PD-1)/programmed cell death ligand 1(PD-L1) have been clinically approved for melanoma, renal cellcarcinoma, non-small lung cancer and advanced metastatic cutaneoussquamous cell carcinoma. Blocking PD-1 releases the break on T-cellactivation, but its efficacy as a single agent often it is notsufficient to get tumor clearance and durable anti-tumor responses.

SUMMARY OF THE INVENTION

The present invention provides an immunotherapeutic modality usingbi-specific antibodies targeted against Cluster of Differentiation 28(CD28) and Epidermal Growth Factor Receptor (EGFRxCD28 or CD28xEGFR)that, when combined with a PD-1 blocking antibody, induce long livedanti-tumor immunity and promote robust intra-tumoral T-cell activationwith no signs of systemic cytokine secretion, in both syngeneic andhuman tumor xenograft models. Toxicology studies ingenetically-humanized immunocompetent mice and in cynomolgus monkeysdemonstrate that these bi-specifics exhibited some single agent activityand no toxicity on their own or in combination with anti-PD-1 antibody.Collectively, these data suggest that combining this class of CD28-basedbi-specifics with PD-1 inhibition may provide safe, biologics solutionswith strikingly enhanced, specific and synergistic anti-tumor activity.

In one aspect, the present invention provides an isolated bispecificantigen binding molecule comprising a first antigen-binding domain thatbinds human CD28 with a K_(D) of less than about 10⁻⁶M as measured bysurface plasmon resonance at 25° C.; and a second antigen-binding domainthat specifically binds a human epidermal growth factor receptor (EGFR)on a target tumor cell, with a K_(D) of less than about 10⁻⁹M asmeasured by surface plasmon resonance at 25° C.

In another aspect, the isolated bispecific antigen binding moleculedemonstrates a costimulatory effect when used in conjunction with ananti-Mucine 16 (MUC16) X CD3 bispecific antibody and tested on targetcells expressing EGFR. In one embodiment, the costimulatory effect isshown by one or more of the following: (a) the ability to activate anddirect human T cells to kill a target cell expressing EGFR; (b) theability to upregulate PD-1 on T cells; (c) the ability to increase therelease of the cytokines IFN gamma and TNF from PBMC; (d) the ability todeplete tumor cells; or (f) the ability to enhance tumor clearance. Inanother embodiment, the costimulatory effect is further shown by one ormore of the following: (g) activation of NFκB activity in a T cell/APCluciferase-based reporter assay; or (h) measurement of IL-2 cytokineproduction using a primary CD4⁺ T cell/APC functional assay.

The present invention provides a multispecific (e.g., bi-specific)antigen-binding protein (e.g., antibody or antigen-binding fragmentthereof) that binds EGFR and CD28 comprising: (1) an EGFR binding armcomprising: (a) an immunoglobulin chain comprising the HCDRs of a heavychain variable region that comprises an amino acid sequence set forth ina member selected from the group consisting of SEQ ID NOs: 2, 30, 40 and50, or a variant thereof; and/or (b) an immunoglobulin chain comprisingthe LCDRs of a light chain variable region that comprises an amino acidsequence set forth in SEQ ID NO: 16, or a variant thereof; and a CD28binding arm; or (2) a CD28 binding arm comprising: (c) an immunoglobulinchain comprising the HCDRs of a heavy chain variable region thatcomprises an amino acid sequence set forth in a member selected from thegroup consisting of SEQ ID NOs: 10, 59 and 63, or a variant thereof;and/or (d) an immunoglobulin chain comprising the LCDRs of a light chainvariable region that comprises an amino acid sequence set forth in amember selected from the group consisting of SEQ ID NOs: 16 and 67, or avariant thereof; and an EGFR binding arm.

For example, in an embodiment of the invention, the multispecific (e.g.,bi-specific) antigen-binding protein (e.g., antibody or antigen-bindingfragment thereof) that binds EGFR and CD28 includes: (1) an EGFR bindingarm comprising: (a) a heavy chain immunoglobulin or variable regionthereof comprising an amino acid sequence having at least 90% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NOs: 2,24, 30, 40 and 50, or a variant thereof; and/or (b) a light chainimmunoglobulin or variable region thereof comprising an amino acidsequence having at least 90% amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NOs: 16 and 28, or a variant thereof;and a CD28 binding arm; or (2) a CD28 binding arm comprising: (c) aheavy chain immunoglobulin or variable region thereof comprising anamino acid sequence having at least 90% amino acid sequence identity tothe amino acid sequence set forth in SEQ ID NOs: 10, 26, 59 and 63, or avariant thereof; and/or (d) a light chain immunoglobulin or variableregion thereof comprising an amino acid sequence having at least 90%amino acid sequence identity to the amino acid sequence set forth in SEQID NOs: 16, 28, and 67, or a variant thereof; and an EGFR binding arm.

In an embodiment of the invention, the multispecific antigen-bindingprotein (e.g., antibody or antigen-binding fragment thereof) that bindsEGFR and CD28 includes: (1) an EGFR binding arm comprising: (a) a heavychain immunoglobulin or variable region thereof comprising the HCDR1,HCDR2 and HCDR3 of a heavy chain variable region comprising an aminoacid sequence set forth in SEQ ID NO: 2, 30, 40 or 50, and at least 90%amino acid sequence identity to the amino acid sequence set forth in SEQID NO: 2, 30, 40 or 50, respectively; and/or a light chainimmunoglobulin or variable region thereof comprising the LCDR1, LCDR2and LCDR3 of a light chain variable region comprising an amino acidsequence set forth in SEQ ID NO: 16, and at least 90% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO: 16,respectively; and a CD28 binding arm; or (2) a CD28 binding armcomprising: (c) a heavy chain immunoglobulin or variable region thereofcomprising the HCDR1, HCDR2 and HCDR3 of a heavy chain variable regioncomprising an amino acid sequence set forth in SEQ ID NO: 10, 59 or 63,and at least 90% amino acid sequence identity to the amino acid sequenceset forth in SEQ ID NO: 10, 59 or 63, respectively; and/or (d) a lightchain immunoglobulin or variable region thereof comprising the LCDR1,LCDR2 and LCDR3 of a light chain variable region comprising an aminoacid sequence set forth in SEQ ID NO: 16, or 67, and at least 90% aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO: 16, or 67, respectively; and an EGFR binding arm.

In an embodiment of the invention, the multispecific (e.g., bi-specific)antigen-binding protein (e.g., antibody or antigen-binding fragmentthereof) that binds EGFR and CD28 includes:

(1)

-   -   an EGFR binding arm that comprises: a CDR-H1 that comprises the        amino acid sequence: GDSIITFY (SEQ ID NO: 4; or a variant        thereof); a CDR-H2 that comprises the amino acid sequence:        IYYSGIT (SEQ ID NO: 6; or a variant thereof); and a CDR-H3 that        comprises the amino acid sequence: ARVSEDSYFHYGMDV (SEQ ID NO:        8; or a variant thereof); and a CDR-L1 that comprises the amino        acid sequence: QSVSSSY (SEQ ID NO: 18; or a variant thereof); a        CDR-L2 that comprises the amino acid sequence: GAS (SEQ ID NO:        20; or a variant thereof); and a CDR-L3 that comprises the amino        acid sequence: QQYGSSPWT (SEQ ID NO: 22; or a variant thereof);        and    -   a CD28 binding arm that comprises:    -   a CDR-H1 that comprises the amino acid sequence: GGSISSYY (SEQ        ID NO: 12; or a variant thereof); a CDR-H2 that comprises the        amino acid sequence: IYYSGIT (SEQ ID NO: 6; or a variant        thereof); and a CDR-H3 that comprises the amino acid sequence:        ARWGVRRDYYYYGMDV (SEQ ID NO: 14; or a variant thereof); and a        CDR-L1 that comprises the amino acid sequence: QSVSSSY (SEQ ID        NO: 18; or a variant thereof);    -   a CDR-L2 that comprises the amino acid sequence: GAS (SEQ ID NO:        20; or a variant thereof);    -   a CDR-L3 that comprises the amino acid sequence: QQYGSSPWT (SEQ        ID NO: 22; or a variant thereof);        (2)    -   an EGFR binding arm that comprises: a CDR-H1 that comprises the        amino acid sequence: GFTFSTFI (SEQ ID NO: 32; or a variant        thereof); a CDR-H2 that comprises the amino acid sequence:        ISSNGGTI (SEQ ID NO: 34; or a variant thereof); and a CDR-H3        that comprises the amino acid sequence: TRGGDFWSGYYPFDY (SEQ ID        NO: 36; or a variant thereof); a CDR-L1 that comprises the amino        acid sequence: QSVSSSY (SEQ ID NO: 18; or a variant thereof);    -   a CDR-L2 that comprises the amino acid sequence: GAS (SEQ ID NO:        20; or a variant thereof); and a CDR-L3 that comprises the amino        acid sequence: QQYGSSPWT (SEQ ID NO: 22; or a variant thereof);        and    -   a CD28 binding arm that comprises: a CDR-H1 that comprises the        amino acid sequence: GGSISSYY (SEQ ID NO: 12; or a variant        thereof); a CDR-H2 that comprises the amino acid sequence:        IYYSGIT (SEQ ID NO: 6; or a variant thereof); a CDR-H3 that        comprises the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO:        14; or a variant thereof);    -   and a CDR-L1 that comprises the amino acid sequence: QSVSSSY        (SEQ ID NO: 18; or a variant thereof); a CDR-L2 that comprises        the amino acid sequence: GAS (SEQ ID NO: 20; or a variant        thereof); and a CDR-L3 that comprises the amino acid sequence:        QQYGSSPWT (SEQ ID NO: 22; or a variant thereof);        (3)    -   an EGFR binding arm that comprises: a CDR-H1 that comprises the        amino acid sequence: GFSFRDAW (SEQ ID NO: 42; or a variant        thereof); a CDR-H2 that comprises the amino acid sequence:        IRNKIDGGTT (SEQ ID NO: 44; or a variant thereof); and a CDR-H3        that comprises the amino acid sequence: TTDIWNYVLFYYYGLDV (SEQ        ID NO: 46; or a variant thereof); and a CDR-L1 that comprises        the amino acid sequence: QSVSSSY (SEQ ID NO: 18; or a variant        thereof); a CDR-L2 that comprises the amino acid sequence: GAS        (SEQ ID NO: 20; or a variant thereof); and a CDR-L3 that        comprises the amino acid sequence: QQYGSSPWT (SEQ ID NO: 22; or        a variant thereof) and    -   a CD28 binding arm that comprises: a CDR-H1 that comprises the        amino acid sequence: GGSISSYY (SEQ ID NO: 12; or a variant        thereof); a CDR-H2 that comprises the amino acid sequence:        IYYSGIT (SEQ ID NO: 6; or a variant thereof); and a CDR-H3 that        comprises the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO:        14; or a variant thereof); and a CDR-L1 that comprises the amino        acid sequence: QSVSSSY (SEQ ID NO: 18; or a variant thereof); a        CDR-L2 that comprises the amino acid sequence: GAS (SEQ ID NO:        20; or a variant thereof); and a CDR-L3 that comprises the amino        acid sequence: QQYGSSPWT (SEQ ID NO: 22; or a variant thereof);        (4)    -   an EGFR binding arm that comprises: a CDR-H1 that comprises the        amino acid sequence: DDSIISYY (SEQ ID NO: 52; or a variant        thereof); a CDR-H2 that comprises the amino acid sequence:        IYYSGRT (SEQ ID NO: 54; or a variant thereof); and a CDR-H3 that        comprises the amino acid sequence: ARVSEDSYYHYGMDV (SEQ ID NO:        56; or a variant thereof); and a CDR-L1 that comprises the amino        acid sequence: QSVSSSY (SEQ ID NO: 18; or a variant thereof); a        CDR-L2 that comprises the amino acid sequence: GAS (SEQ ID NO:        20; or a variant thereof); and a CDR-L3 that comprises the amino        acid sequence: QQYGSSPWT (SEQ ID NO: 22; or a variant thereof);        and    -   a CD28 binding arm that comprises: a CDR-H1 that comprises the        amino acid sequence: GGSISSYY (SEQ ID NO: 12; or a variant        thereof); a CDR-H2 that comprises the amino acid sequence:        IYYSGIT (SEQ ID NO: 6; or a variant thereof); and a CDR-H3 that        comprises the amino acid sequence: ARWGVRRDYYYYGMDV (SEQ ID NO:        14; or a variant thereof); and a CDR-L1 that comprises the amino        acid sequence: QSVSSSY (SEQ ID NO: 18; or a variant thereof); a        CDR-L2 that comprises the amino acid sequence: GAS (SEQ ID NO:        20; or a variant thereof); and a CDR-L3 that comprises the amino        acid sequence: QQYGSSPWT (SEQ ID NO: 22; or a variant thereof).

In an embodiment of the invention, the multispecific (e.g., bi-specific)antigen-binding protein (e.g., antibody or antigen-binding fragmentthereof) that binds EGFR and CD28 includes:

(1)an EGFR binding arm that comprises: a heavy chain variable region thatcomprises the amino acid sequence set forth in SEQ ID NO: 2 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof);and a CD28 binding arm that comprises: a heavy chain variable regionthat comprises the amino acid sequence set forth in SEQ ID NO: 10 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof);(2)an EGFR binding arm that comprises: a heavy chain variable region thatcomprises the amino acid sequence set forth in SEQ ID NO: 30 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof);and a CD28 binding arm that comprises: a heavy chain variable regionthat comprises the amino acid sequence set forth in SEQ ID NO: 10 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof);(3)an EGFR binding arm that comprises: a heavy chain variable region thatcomprises the amino acid sequence set forth in SEQ ID NO: 40 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof);and a CD28 binding arm that comprises: a heavy chain variable regionthat comprises the amino acid sequence set forth in SEQ ID NO: 10 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof);(4)an EGFR binding arm that comprises: a heavy chain variable region thatcomprises the amino acid sequence set forth in SEQ ID NO: 50 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof);and a CD28 binding arm that comprises: a heavy chain variable regionthat comprises the amino acid sequence set forth in SEQ ID NO: 10 (or avariant thereof); and a light chain variable region that comprises theamino acid sequence set forth in SEQ ID NO: 16 (or a variant thereof).

In an embodiment of the invention, the multispecific (e.g., bi-specific)antigen-binding protein (e.g., antibody or antigen-binding fragmentthereof) that binds EGFR and CD28 includes:

(1)an EGFR binding arm that comprises: a heavy chain that comprises theamino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof);and a light chain that comprises the amino acid sequence set forth inSEQ ID NO: 28 (or a variant thereof); and a CD28 binding arm thatcomprises: a heavy chain that comprises the amino acid sequence setforth in SEQ ID NO: 26 (or a variant thereof); and a light chain thatcomprises the amino acid sequence set forth in SEQ ID NO: 28 (or avariant thereof);(2)an EGFR binding arm that comprises: a heavy chain that comprises theamino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof);and a light chain that comprises the amino acid sequence set forth inSEQ ID NO: 28 (or a variant thereof); and a CD28 binding arm thatcomprises: a heavy chain that comprises the amino acid sequence setforth in SEQ ID NO: 26 (or a variant thereof); and a light chain thatcomprises the amino acid sequence set forth in SEQ ID NO: 28 (or avariant thereof);(3)an EGFR binding arm that comprises: a heavy chain that comprises theamino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof);and a light chain that comprises the amino acid sequence set forth inSEQ ID NO: 28 (or a variant thereof);anda CD28 binding arm that comprises: a heavy chain that comprises theamino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof);and a light chain that comprises the amino acid sequence set forth inSEQ ID NO: 28 (or a variant thereof);(4)an EGFR binding arm that comprises: a heavy chain that comprises theamino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof);and a light chain that comprises the amino acid sequence set forth inSEQ ID NO: 28 (or a variant thereof);anda CD28 binding arm that comprises: a heavy chain that comprises theamino acid sequence set forth in SEQ ID NO: 26 (or a variant thereof);and a light chain that comprises the amino acid sequence set forth inSEQ ID NO: 28 (or a variant thereof).

In an embodiment of the invention, the multispecific (e.g., bispecific)antigen-binding protein (e.g., an antibody or antigen-binding fragmentthereof) is REGN7075 (also referred to herein as bsAb7075); REGN6321(also referred to herein as bsAb6321); REGN6322 (also referred to hereinas bsAb6322); REGN6323 (also referred to herein as bsAb6323). Otherbispecific antibodies may be generated using the parental EGFRantibodies described herein in Tables 9A, 9B and 9C, whereby thesequences of the HCVR arms of the EGFR parental antibodies may be foundin WO2014/004427 and the HCVR amino acid sequences of the parental CD28antibodies may be found in Table 3 herein. Any EGFR HCVR arm may bepaired with any of the CD28 HCVR arms described herein.

The present invention also provides an antigen-binding protein (e.g.,antibody or antigen-binding fragment thereof) that specifically binds toepidermal growth factor receptor comprising: (i) a heavy chainimmunoglobulin or variable region thereof that comprises HCDR1, HCDR2and HCDR3 of a heavy chain immunoglobulin or variable region thereofthat comprises the amino acid sequence set forth in SEQ ID NO: 2, 24,30, 38, 40, 48, 50 or 58; or a variant thereof; and/or (ii) a lightchain immunoglobulin or variable region thereof that comprises LCDR1,LCDR2 and LCDR3 of a light chain immunoglobulin or variable regionthereof that comprises the amino acid sequence set forth in SEQ ID NO:16 or 28; or a variant thereof. In an embodiment of the invention, theantigen-binding protein is multispecific (e.g., bispecific).

The present invention also provides a multispecific antigen-bindingprotein set forth herein bound to an EGFR polypeptide or an antigenicfragment thereof (e.g., on the surface of a tumor cell) and a CD28polypeptide or an antigenic fragment thereof (e.g., on the surface of animmune cells such as a T-cell).

The present invention also provides a method for making a multispecific(e.g., bi-specific) antigen-binding protein set forth herein comprising:(a) introducing one or more polynucleotides encoding the immunoglobulinchains of said antigen-binding protein into a host cell (e.g., a CHOcell); (b) culturing the host cell under conditions favorable toexpression of the polynucleotide; and (c) optionally, isolating theantigen-binding protein or immunoglobulin chain from the host celland/or medium in which the host cell is grown. Any antigen-bindingprotein or immunoglobulin chain which is a product of such a method ispart of the present invention.

The present invention also provides a polynucleotide encoding one ormore of the immunoglobulin chains of a multispecific (e.g., bi-specific)antigen-binding protein of the present invention. Vectors comprising thepolynucleotides of the present invention are also part of the presentinvention as well as host cells (e.g., CHO) comprising a polynucleotide,vector or antigen-binding protein of the present invention.

The present invention also provides a composition or kit comprising oneor more of the multispecific antigen-binding proteins of the presentinvention in association with one or more other substances or items,optionally, in association with a further therapeutic agent (e.g., PD-1inhibitor, an anti-PD-1 antibody or antigen-binding fragment thereof, aPD-L1 inhibitor, an anti-PD-L1 antibody or antigen-binding fragmentthereof, a platinum anti-cancer chemotherapeutic agent, paclitaxel,docetaxel, vincristine, cisplatin, carboplatin, oxaliplatin, ananti-cancer antibody or antigen-binding fragment thereof, pembrolizumab,nivolumab, trastuzumab, cetuximab, bevacizumab and/or cemiplimab).Pharmaceutical formulations comprising the multispecific antigen-bindingprotein of the present invention and a pharmaceutically acceptablecarrier or excipient and, optionally, a further therapeutic agent arealso part of the present invention.

The present invention also provides vessels and injection devices (e.g.,syringe, pre-filled syringe or autoinjector) including the multispecificantigen-binding proteins, compositions and/or formulations of thepresent invention.

The present invention also provides a method for administering amultispecific antigen-binding protein of the present invention,composition or formulation thereof to a subject (e.g. a human)comprising injecting said antigen-binding protein, composition orformulation into the body of the subject, e.g., with a syringe,pre-filled syringe or autoinjector. Optionally a further therapeuticagent (e.g., cemiplimab, pembrolizumab or nivolumab) is alsoadministered to the subject.

The present invention also provides a method for treating or preventinga hyperproliferative disease (e.g., EGFR-expressing cancer), in asubject (e.g., a human) in need thereof, comprising administering (e.g.,subcutaneously, intravenously or intramuscularly) an effective amount ofmultispecific antigen-binding protein or composition or formulation,optionally in association with a further therapeutic agent (e.g.,cemiplimab, pembrolizumab or nivolumab). In an embodiment of theinvention, the EGFR-expressing cancer is esophageal carcinoma, lungsquamous cell carcinoma, lung adenocarcinoma, cervical squamous cellcarcinoma, endometrial adenocarcinoma, bladder urothelial carcinoma,lung cancer, non-small cell lung cancer, colorectal cancer, rectalcancer, endometrial cancer, skin cancer, head & neck squamous cellcarcinoma, brain cancer, glioblastoma multiforme, breast cancer,gastroesophageal cancer, gastroesophageal adenocarcinoma, prostatecancer or ovarian cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, and 1E show that, in cancer cell lines withendogenous TSA, EGFRxCD28 bispecific antibody (REGN7075) potentiatesT-cell activation in the presence of TCR stimulation by TSAxCD3(MUC16xCD3). In FIGS. 1A and 1B, flow cytometry analysis shows thatEGFRxCD28 bispecific antibody binds to CD28+ and EGFR+ cells. (FIG. 1A)Jurkat cells (CD28+ cells). (FIG. 1B) PEO1 cells (EGFR+ cells). In FIGS.1C-1E, human T-cells were cultured with cancer target cells expressingendogenous MUC16 and EGFR (Ovarian cancer cell line PEO1) and theindicated bispecifics for 96 hours. (FIG. 1C) Tumor cell kill, % viablecells. (FIG. 1D) frequency of CD25⁺ T cells (% of CD2). (FIG. 1E)Supernatants from cytotoxicity assay were analyzed using a CytometricBead Array (CBA) kit. IFNγ released is plotted as pg/ml

FIGS. 2A, 2B, 2C, and 2D show that expression of a CD28 ligand (CD86) ontumor cells synergizes with anti-PD1 treatment to induce CD8 dependentanti-tumor immunity. MC38 tumor cells were transduced with the ligandfor CD28, CD86 (MC38/CD86), or empty vector control (MC38/EV). WT C57BL6mice were initially implanted with 1×10⁶ tumor cells per mouse andtreated with PD-1 mAb or rIgG2a isotype control at 5 mg/kg on day 0, 3,7, 10 and 14 post tumor implant. FIG. 2A. Average tumor volume overtime. Error bars represent +/−SEM. Statistical significance wasdetermined with two-way ANOVA and Tukey's multiple comparisons tests.FIG. 2B. Survival over time (percentage of mice with tumors <2000 mm³).Statistical significance at day 60 post-implantation was determined withthe Log-rank (Mantel-Cox) test. FIG. 2C. Mice were treated CD8 depletingantibody (CD8 depleted) or isotype control (no depletion). Average tumorvolume over time with CD8 depletion (dotted lines) and no depletion(solid lines) is shown +/−SEM. Statistical significance was determinedwith two-way ANOVA and Tukey's multiple comparisons tests. FIG. 2D.Secondary tumor implant (re-challenge) of tumor free mice that wereimplanted with MC38/CD86 and treated with PD1 mAb. For FIGS. 2A to 2D,data are shown from 1 experiment with 10 mice per group. Data arerepresentative of at least 4 separate experiments. Statisticalsignificance is indicated (*p<0.05, **p<0.01, ***p<0.001, and****p<0.0001).

FIGS. 3A and 3B show A431 Human xenograft tumor model. Data correspondswith FIGS. 4 and 5 . FIG. 3A shows human CD45⁺ cell engraftment inperipheral blood, pre-treatment. FIG. 3B shows A431 FACS analysis.Marker of interest is indicated for each plot.

FIG. 4 and FIG. 5 show EGFRxCD28 (REGN7075) synergizes with anti-PD1(cemiplimab) treatment to induce anti-tumor immunity in VH miceengrafted with human fetal liver CD34⁺ cells. Specifically, in FIGS. 4and 5 , A431 epidermoid carcinoma tumor cells (obtained from ATCC) wereimplanted subcutaneously in SIRPA^(h/h) TPO^(h/m) Rag2^(−/−) II2rg^(−/−)mice that were engrafted with fetal liver CD34⁺ cells. Mice weresegregated into indicated treatment groups based on fetal liver donor,human immune cell engraftment frequency and sex. Mice were treatedintra-peritoneally (IP) starting on the day of tumor challenge andadministrated every 2-3 days through the experiment. Dose for anti-PD-1is 10 mg/kg and EGFRxCD28 or PSMAxCD28, 5 mg/kg. Data shown are averageA431 tumor volumes for each treatment group (mm³±SEM) plotted againstdays after tumor challenge. Two-way ANONA, Tukey comparison: ** P<0.01,**** P<0.001. (n=10˜12 mice per group).

FIG. 4 specifically shows average A431 tumor volumes for each treatmentgroup (mm³±SEM) (isotype, anti-PD1 monotherapy (cemiplimab), EGFRxCD28(REGN7075) monotherapy or EGFRxCD28 (REGN7075)+anti-PD1 combinationtherapy) plotted against days after tumor in VH mice engrafted withhuman CD34⁺ cells. Two-way ANONA, Tukey comparison: ** P<0.01, ****P<0.0001.

FIG. 5 shows average A431 tumor volumes for control group (mm³±SEM)(isotype, anti-PD1 monotherapy (cemiplimab), PSMAxCD28 monotherapy orPSMAxCD28+anti-PD1 combination therapy) plotted against days after tumorin VH mice engrafted with human CD34⁺ cells. Two-way ANONA, Tukeycomparison: ** P<0.01, **** P<0.0001.

FIG. 6 shows the validation of the lack of PSMA expression on A431 tumorcells. MC38 mouse tumor cell line engineered to express human PSMA wasused as positive control.

FIGS. 7A and 7B. Average A431 tumor volumes for each treatment group(mm³±SEM) receiving EGFRxCD28 (REGN7075) plotted against days aftertumor challenge in NSG mice either prophylactically (FIG. 7A) ortherapeutically (FIG. 7B) treated. Two-way ANONA, Tukey comparison: ****P<0.0001.

FIG. 8 . Average A549 tumor volumes for each treatment group (mm³±SEM)(isotype or EGFRxCD28 (REGN7075)@ 1 mg/kg or 10 mg/kg) plotted againstdays after tumor challenge in NSG mice. Two-way ANONA, Tukeycomparison: * isotype vs EGFRxCD28 1 mg/kg (P<0.05), ****: P<0.0001; $isotype vs EGFRxCD28 10 mg/kg (P<0.05), $$$: P<0.001, $$$$: P<0.0001; @EGFRxCD28 1 mg/kg vs 10 mg/kg (P<0.05).

FIGS. 9A, 9B, 9C, and 9D. EGFRxCD28 (REGN7075) synergizes with anti-PD1(cemiplimab) treatment to induce anti-tumor immunity in VH miceengrafted with human fetal liver CD34⁺ cells. (FIG. 9A) MFI of selectedtumor CD8⁺ T-cell clusters identified by CITRUS (cluster identification,characterization, and regression) analysis; (FIG. 9B) Frequency of cellsin each cluster from the indicated treatment groups; (FIG. 9C) MFI ofselected tumor CD4+ T-cell clusters identified by CITRUS analysis; (FIG.9D) Frequency of cells in each cluster from the indicated treatmentgroups. FIGS. 9B and 9D demonstrate that EGFRxCD28 synergizes withanti-PD1 treatment to induce anti-tumor immunity in VH mice engraftedwith human fetal liver CD34⁺ cells.

FIGS. 10A, 10B, and 10C. EGFRxCD28 (REGN7075) alone or in combinationwith anti-PD1 (cemiplimab) therapy does not induce systemic T-cellactivation in comparison to CD28 superagonist in cynomolgus monkeys.Cynomolgus monkeys were treated with a single dose of bi-specificantibodies at the indicated dose (mg/kg). Time is indicated post-dose(hr) (FIG. 10A) Serum cytokines (IFNgamma, IL-2, IL-6, IL-8 and IL-10)(FIG. 10B) Relative peripheral blood T-cell counts (FIG. 10C) Frequencyof Ki67⁺ and ICOS+ T-cells (% of CD3). Values represent the average+/−SEM. N=3 animals per group. P values were calculated with 2way ANOVAwith comparison to isotype control. (**, p<0.01; ***, p<0.001 and ****,p<0.0001).

FIG. 11 shows a dose dependent anti-tumor response over time which ismediated by an anti-EGFR X anti-CD28 antibody (REGN7075) when combinedwith a PD-1 antibody (REGN2810, described as H2M7798N or H4H7798N inUS20150203579).

FIG. 12 shows a dose dependent anti-tumor response mediated by ananti-EGFR X anti-CD28 antibody (REGN7075) when combined with a PD-1antibody (REGN2810).

FIGS. 13A, 13B, and 13C describe the binding of various anti-EGFR Xanti-CD28 antibodies to various cells, including human and cynomolgus Tcells and Jurkat cells (CD28+effector cells)(FIG. 13A); various othertumor target cells including A375 melanoma cells, 22RV1 prostate cells,PEO1 ovarian cells, CAPAN pancreatic cells, SW1990 Pancreatic cells andH292 lung cells (FIGS. 13B and 13C). Isotype Control I and II areantibodies that bind to Mers and EGFRvIII, respectively.

FIGS. 14A and 14B show that an anti-EGFR X anti-CD28 bispecific antibodyenhances the cytotoxic potency of an anti-tumor specific antigen (TSA) Xanti-CD3 bispecific antibody. The study included an anti-EGFR Xanti-CD28 antibody combined with either an anti-STEAP2 X anti-CD3bispecific antibody (FIG. 14A) or an anti-PSMA X anti-CD3 bispecificantibody (FIG. 14B). The anti-STEAP2xanti-CD3 antibody was described inWO2018/058001A. The anti-PSMAxanti-CD3 antibody was described inWO2017/053856A1.

FIGS. 15A, 15B, 15C and 15D show that an anti-EGFR X anti-CD28bispecific antibody enhances the cytotoxic potency of an anti-tumorspecific antigen (TSA) X anti-CD3 bispecific antibody (anti-MUC16 Xanti-CD3) across various cell lines, including PEO1 cells (FIG. 15A),CAPAN2 cells (FIG. 15B), SW1990 cells (FIG. 15C) and H292 cells (FIG.15D).

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and1 01 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Checkpoint inhibition with PD-1 blocking mAb are known to release thebreak on T cell activation, but their efficacy as a single agent oftennot always sufficient to get tumor clearance and a durable anti-tumorresponse in many cancers. Several approaches to improve the responserate to PD-1 inhibition are currently being evaluated. Indeed,identification of biomarkers to predict responsiveness to PD-1 mAbs,non-tumor targeted combination therapies using PD-1 inhibition togetherwith agonistic antibodies triggering costimulatory receptors to improveT cell activation, or with chemotherapy or radiotherapy are allcurrently undergoing pre-clinical and clinical testing. The challengehowever is that many of these combinations are often based on theavailability of pre-existing drug and a post-hoc rationale to combinetherapies, rather than a truly hypothesis-driven approach, which in somecases has led to worse outcomes for the patient. It is evident thatcheckpoint inhibition and reactivation of the immune system offers thepotential of long term remission in a number of patients, thereforemethods to further improve or enhance T cell activity to promote a moredurable responses are warranted. Here, to improve the anti-tumorefficacy of PD-1 mAb, the concept of using an EGFRxCD28 bispecific toenhance T-cell signaling and activation was introduced. Indeed, thisnovel combination immunotherapy demonstrated that CD28 costimulatorybispecific antibodies synergize with PD-1 mAb to not only generaterobust T-cell activation but also to provide durable anti-tumorresponses without systemic toxicity. Consequently, this tumor-targetedcombination therapy provides a considerable advantage over thenon-targeted approaches described previously. Using CD28-bispecificantibodies, which do not directly activate CD28 unless clustered ontumor cell surfaces, offered the possibility of promoting co-stimulationonly at the tumor site, avoiding the systemic toxicity of conventionalCD28-activating antibodies, the toxicity often observed with thecombination of CLTA-4 and PD-1 blockade or other costimulatory agonistbivalent antibodies. Toxicology studies in genetically-humanizedimmunocompetent mice, as well as in cynomolgus monkeys, showed thatthese bispecifics exhibited no toxicity as single agents or incombination with PD-1 mAb. The safety profile together with the similarenhancement of anti-tumor efficacy by EGFRxCD28 with anti-PD-1 mAbacross syngeneic and xenogeneic models suggested that this therapeuticmodality is robust, not limited to a specific tumor model, and couldhave broader utility as a novel combination class for immunotherapy.

To enhance T-cell-mediated killing of tumor cells, tumor-targetedapproaches are being developed. Indeed, CD3-based bispecific antibodiesrepresent an emerging class of antibodies that can efficiently triggerT-cell activation, by linking a T cell to a tumor cell and activatingTCR/CD3, thus mimicking normal “signal 1”. However, despite theirpromising clinical efficacy, CD3-bispecifics can be associated withcytokine release syndrome (CRS) due to direct T-cell activation and lackof tumor only specificity. Further, TCR/CD3 activation in the absence ofco-stimulation (“signal 2”) can lead to anergy or activation inducedcell death (AICD), which may limit or reduce the potential anti-tumoreffects of these reagents. Here it was demonstrated for the first timethat EGFRxCD28 bispecific and anti-PD-1 mAb combination therapy induceda tumor specific T-cell activation. As shown herein, EGFRxCD28bispecific antibodies have limited or no activity in the absence of“signal 1” and PD-1 blockade relies on the endogenous antigen specificT-cell response to tumor peptides. Thus, CD28-bispecifics together withPD-1 blockade can boost endogenous TCR/CD3-dependent T cell responsesdriving durable anti-tumor responses.

The anti-tumor activity of PD-1 mAb is CD28-dependent and the PD-1inhibition of T-cell activation reduces signaling through TCR/CD3 and/orCD28 which may be affecting the spatial localization of those molecules.The data herein demonstrated that PD-1 is accumulated at the immunesynapse when PD-L1 is expressed by target cells and its accumulation isassociated with a reduction of CD28 at the synapse, suggesting that PD-1could exercise T-cell inhibition, by preventing CD28 localization to thesynapse. In addition, it was found that PD-1 blockade prevented PD-1synaptic localization while CD28 accumulation at the synapse wasincreased, allowing EGFRxCD28 bispecifics to markedly enhance theability of PD-1 mAb to promote T-cell activation. This may be one of themechanisms by which PD-1 blocking antibody promotes T-cell activation.Overall, the visualization of PD-1 and CD28 localization in theimmunological synapse following PD-1-PD-L1 interaction and/or PD-1inhibition, made it possible to better understand the effect of PD-1blockade on T-cell activation, as well as the synergy between EGFRxCD28and PD-1 mAb at the level of the immune synapse.

Although PD-1 mAbs are an important new class of immunotherapy, furtheroptimization of anti-tumor activity will surely be necessary in manycases. Just as CAR-T approaches have employed chimeric receptors thatartificially activate both “signal 1” and “signal 2” so as to improvetheir anti-tumor activity, it is now shown the potential benefit ofcombining PD-1 inhibition with CD28-bispecifics (which provide “signal2”) to enhance anti-tumor activity. This approach has several practicalbenefits over CAR-T therapies in that it does not require a laboriouscell therapy preparation that must be individually customized for eachpatient, nor does it require that patients be pre-emptively“lymphodepleted” via toxic chemotherapy that is often associated withadverse effects so that they can't accept cell therapy. This bispecificapproach offers the potential for increased efficacy as well asincreased safety through its specificity of action. Collectively, thedata here suggest that combining CD28-based bispecifics with theclinically validated PD-1 mAb, such as cemiplimab, may providewell-tolerated, biologics solutions with markedly enhanced andsynergistic anti-tumor activity.

Definitions

“EGFR” and “EGFR fragment,” as used herein refer to the well-known humanEGFR protein or a fragment thereof unless specified as being from anon-human species (e.g., “mouse EGFR,” “mouse EGFR fragment,” “monkeyEGFR,” “monkey EGFR fragment,” etc.). In an embodiment of the invention,human EGFR comprises the amino acid sequence set forth in NCBI accessionno. NP_005219.2. In one embodiment, human EGFR (amino acids L25-A647 ofAccession number 005228.4) is shown with a C-terminal CPGG.myc epitope(E1-L10).GlyGly.myc epitope (E1-L10).SerGly.6XHis.SSG tag (SEQ ID NO:69).

“CD28,” as used herein, refers to the well-known human CD28 proteinwhich is express on T cells as a costimulatory receptor unless specifiedas being from a non-human species. In an embodiment of the invention,human CD28 comprises the amino acid sequence as set forth NCBI accessionNo. NP_006130.1.

“Isolated” antigen-binding proteins (e.g., antibodies or antigen-bindingfragments thereof), polypeptides, polynucleotides and vectors, are atleast partially free of other biological molecules from the cells orcell culture from which they are produced. Such biological moleculesinclude nucleic acids, proteins, other antibodies or antigen-bindingfragments, lipids, carbohydrates, or other material such as cellulardebris and growth medium. An isolated antigen-binding protein mayfurther be at least partially free of expression system components suchas biological molecules from a host cell or of the growth mediumthereof. Generally, the term “isolated” is not intended to refer to acomplete absence of such biological molecules or to an absence of water,buffers, or salts or to components of a pharmaceutical formulation thatincludes the antigen-binding proteins (e.g., antibodies orantigen-binding fragments).

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J.272(20): 5101-5109; Altschul, S. F., et aL., (1990) J. Mol. Biol.215:403-410; Gish, W., et aL., (1993) Nature Genet. 3:266-272; Madden,T. L., et aL., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., etaL., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et aL., (1997)Genome Res. 7:649-656; Wootton, J. C., et aL., (1993) Comput. Chem.17:149-163; Hancock, J. M. et aL., (1994) Comput. Appl. Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et aL., “A model ofevolutionary change in proteins.” in Atlas of Protein Sequence andStructure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352,Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et aL.,“Matrices for detecting distant relationships.” in Atlas of ProteinSequence and Structure, (1978) vol. 5, suppl. 3.″ M. O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et aL., (1991)Methods 3:66-70; Henikoff, S., et aL., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et aL., (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268; Karlin, S., et aL., (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877; Dembo, A., et aL., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, N.Y.

The present invention includes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 6; 8; 10; 12; 14; 24; 34; 36; 38; 44;46; 48; 54; 56 and/or 58.

An “antibody” is an immunoglobulin molecule comprising four polypeptidechains, two heavy chains (HC) and two light chains (LC) inter-connectedby disulfide bonds. Each heavy chain (HC) comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region (e.g., IgG, IgG1 or IgG4). The heavy chain constantregion comprises three domains, C_(H)1, C_(H)2 and C_(H)3. Each lightchain (LC) comprises a light chain variable region (abbreviated hereinas LCVR or V_(L)) and a light chain constant region (e.g., lambda orkappa). The light chain constant region comprises one domain (C_(L)1).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) includes three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A heavy chain CDR may be referredto as HCDR and a light chain CDR may be referred to as LCDR. Indifferent embodiments of the invention, the FRs of an antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified.

An antigen-binding arm of a Y-shaped IgG antibody (e.g., a CD28 or EGFRbinding arm) refers to a structural portion of the antibody that confersbinding specificity to the antigen. For example, an antigen-binding armof an IgG antibody has a heavy chain (HC) associated with a light chain(LC).

An antibody which, for example, is bispecific includes an arm that bindsto a first antigen and another arm that binds to a second antigen. Forexample, an EGFRxCD28 bispecific antibody includes one arm that bindsEGFR and another arm that binds to CD28.

Bispecific antigen-binding molecules (e.g., bispecific antibodies) mayhave an effector arm that binds to a first antigen and a targeting armthat binds to second antigen. The effector arm may be the firstantigen-binding domain (e.g., anti-CD28) that binds to the antigens oneffector cells (e.g., T cells). The targeting arm may be the secondantigen binding domain (e.g., anti-EGFR antibody) that binds to theantigens on target cells (e.g., tumor cells). According to certainexemplary embodiments, the effector arm binds to CD28 and the targetingarm binds to EGFR.

An “antigen-binding portion” of an antibody, “antigen-binding fragment”of an antibody, and the like, as used herein, include any naturallyoccurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. A multispecific antigen-binding fragment ofan antibody binds to multiple antigens (e.g., two different antigens ifthe fragment is bispecific). Antigen-binding fragments of an antibodymay be derived, e.g., from full antibody molecules using any suitablestandard techniques such as proteolytic digestion or recombinant geneticengineering techniques involving the manipulation and expression of DNAencoding antibody variable and optionally constant domains. Non-limitingexamples of antigen-binding fragments include: (i) Fab fragments; (ii)F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v)single-chain Fv (scFv) molecules; and (vi) dAb fragments.

An antigen-binding fragment of an antibody will, in an embodiment of theinvention, comprise at least one variable domain. The variable domainmay be of any size or amino acid composition and will generally compriseat least one CDR, which is adjacent to or in frame with one or moreframework sequences. In antigen-binding fragments having a V_(H) domainassociated with a V_(L) domain, the V_(H) and V_(L) domains may besituated relative to one another in any suitable arrangement. Forexample, the variable region may be dimeric and contain V_(H)-V_(H),V_(H)-V_(L) or V_(L)-V_(L) dimers. Alternatively, the antigen-bindingfragment of an antibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)—C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H) ²; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

The term “recombinant” antigen-binding proteins, such as antibodies orantigen-binding fragments thereof, refers to such molecules created,expressed, isolated or obtained by technologies or methods known in theart as recombinant DNA technology which include, e.g., DNA splicing andtransgenic expression. The term includes antibodies expressed in anon-human mammal (including transgenic non-human mammals, e.g.,transgenic mice), or a host cell (e.g., Chinese hamster ovary (CHO)cell) or cellular expression system or isolated from a recombinantcombinatorial human antibody library. The present invention includesrecombinant antigen-binding proteins as set forth herein.

The term “specifically binds” or “binds specifically” refers to thoseantigen-binding proteins (e.g., antibodies or antigen-binding fragmentsthereof) having a binding affinity to an antigen, such as EGFR or CD28protein, expressed as K_(D), of less than about 10⁻⁶ M (e.g., 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹² M), as measured by real-time,label free bio-layer interferometry assay, for example, at 25° C. or 37°C., e.g., an Octet® HTX biosensor, or by surface plasmon resonance,e.g., BIACORE™, or by solution-affinity ELISA. “Anti-EGFR” refers to anantigen-binding protein (or other molecule such as an antigen-bindingarm), for example an antibody or antigen-binding fragment thereof, thatbinds specifically to EGFR and “anti-CD28” refers to an antigen-bindingprotein (or other molecule such as an antigen-binding arm), for examplean antibody or antigen-binding fragment thereof, that binds specificallyto CD28. “EGFRxCD28” refers to refers to an antigen-binding protein (orother molecule), for example an antibody or antigen-binding fragmentthereof, that binds specifically to EGFR and to CD28 (and, optionally,to one or more other antigens).

The present invention includes antigen-binding proteins, e.g.,antibodies or antigen-binding fragments, that bind to the same EGFR andCD28 epitopes as an antigen-binding protein of the present invention(e.g. REGN7075 (also referred to herein as bsAb7075); REGN6321 (alsoreferred to herein as bsAb6321); REGN6322 (also referred to herein asbsAb6322); REGN6323 (also referred to herein as bsAb6323). Otheranti-EGFR X anti-CD28 antigen-binding proteins may be found in Tables9A, 9B and 9C. The amino acid sequences of the EGFR HCVR arms of thebispecific antibodies described herein may be found in Table 1, whereasthe amino acid sequences of the CD28 HCVR arms of the bispecificantibodies described herein may be found in Table 3. Other EGFR parentalantibodies for use in the present invention are described inWO2014/004427. The amino acid sequences for the EGFR HCVR arm and theCD28 HCVR arm of REGN7075, REGN6321, REGN6322 and REGN6323 are found inTable 6. The amino acid sequences of the common light chain variableregion described in the present invention are also found in Table 6.

The term “epitope” refers to an antigenic determinant (e.g., on EGFR orCD28) that interacts with a specific antigen-binding site of anantigen-binding protein, e.g., a variable region of an antibodymolecule, known as a paratope. A single antigen may have more than oneepitope. Thus, different antibodies may bind to different areas on anantigen and may have different biological effects. The term “epitope”may also refer to a site on an antigen to which B and/or T cells respondand/or to a region of an antigen that is bound by an antibody. Epitopesmay be defined as structural or functional. Functional epitopes aregenerally a subset of the structural epitopes and have those residuesthat directly contribute to the affinity of the interaction. Epitopesmay be linear or conformational, that is, composed of non-linear aminoacids. In certain embodiments, epitopes may include determinants thatare chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics.

Methods for determining the epitope of an antigen-binding protein, e.g.,antibody or fragment or polypeptide, include alanine scanning mutationalanalysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies and NMRanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Prot. Sci. 9: 487-496). Another method that can be used toidentify the amino acids within a polypeptide with which anantigen-binding protein (e.g., antibody or fragment or polypeptide)interacts is hydrogen/deuterium exchange detected by mass spectrometry.See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen andSmith (2001) Anal. Chem. 73: 256A-265A.

The present invention includes antigen-binding proteins that compete forbinding to CD28 and EGFR with an antigen-binding protein of the presentinvention, e.g., (e.g. REGN7075 (also referred to herein as bsAb7075);REGN6321 (also referred to herein as bsAb6321); REGN6322 (also referredto herein as bsAb6322); REGN6323 (also referred to herein as bsAb6323),as well as those described in Tables 9A, 9B and 9C. The term “competes”as used herein, refers to an antigen-binding protein (e.g., antibody orantigen-binding fragment thereof) that binds to an antigen and inhibitsor blocks the binding of another antigen-binding protein (e.g., antibodyor antigen-binding fragment thereof) to the antigen. Unless otherwisestated, the term also includes competition between two antigen-bindingproteins e.g., antibodies, in both orientations, i.e., a first antibodythat binds antigen and blocks binding by a second antibody and viceversa. Thus, in an embodiment of the invention, competition occurs inone such orientation. In certain embodiments, the first antigen-bindingprotein (e.g., antibody) and second antigen-binding protein (e.g.,antibody) may bind to the same epitope. Alternatively, the first andsecond antigen-binding proteins (e.g., antibodies) may bind todifferent, but, for example, overlapping or non-overlapping epitopes,wherein binding of one inhibits or blocks the binding of the secondantibody, e.g., via steric hindrance. Competition betweenantigen-binding proteins (e.g., antibodies) may be measured by methodsknown in the art, for example, by a real-time, label-free bio-layerinterferometry assay. Also, binding competition between antigen-bindingproteins (e.g., monoclonal antibodies (mAbs)) can be determined using areal time, label-free bio-layer interferometry assay on an Octet RED384biosensor (Pall ForteBio Corp.).

Typically, an antibody or antigen-binding fragment of the inventionwhich is modified in some way retains the ability to specifically bindto EGFR and CD28, e.g., retains at least 10% of its EGFR and CD28binding activity (when compared to the parental antibody) when thatactivity is expressed on a molar basis. Preferably, an antibody orantigen-binding fragment of the invention retains at least 20%, 50%,70%, 80%, 90%, 95% or 100% or more of the EGFR and CD28 binding affinityas the parental antibody. It is also intended that an antibody orantigen-binding fragment of the invention may include conservative ornon-conservative amino acid substitutions (referred to as “conservativevariants” or “function conserved variants” of the antibody) that do notsubstantially alter its biologic activity.

A “variant” of a polypeptide, such as an immunoglobulin chain (e.g.,REGN7075 (also referred to herein as bsAb7075); REGN6321 (also referredto herein as bsAb6321); REGN6322 (also referred to herein as bsAb6322);REGN6323 (also referred to herein as bsAb6323) V_(H), V_(L), HC or LC orCDR thereof comprising the amino acid sequence specifically set forthherein), refers to a polypeptide comprising an amino acid sequence thatis at least about 70-99.9% (e.g., at least 70, 72, 74, 75, 76, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 99.5 or 99.9%) identical or similar to a referenced amino acidsequence that is set forth herein (e.g., any of SEQ ID NOs: 6; 8; 10;12; 14; 16; 18; 20; 22; 24; 26; 28; 30; 32; 34; 36; 38; 40; 42; 44; 46;48; 50; 52; 54; 56; or 58-67); when the comparison is performed by aBLAST algorithm wherein the parameters of the algorithm are selected togive the largest match between the respective sequences over the entirelength of the respective reference sequences (e.g., expect threshold:10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gapcosts: existence 11, extension 1; conditional compositional score matrixadjustment).

Moreover, a variant of a polypeptide may include a polypeptide such asan immunoglobulin chain (e.g., REGN7075 (also referred to herein asbsAb7075); REGN6321 (also referred to herein as bsAb6321); REGN6322(also referred to herein as bsAb6322); REGN6323 (also referred to hereinas bsAb6323) V_(H), V_(L), HC or LC or CDR thereof) which may includethe amino acid sequence of the reference polypeptide whose amino acidsequence is specifically set forth herein but for one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations, e.g., one or more missensemutations (e.g., conservative substitutions), non-sense mutations,deletions, or insertions. For example, the present invention includesCD28xEGFR antigen-binding proteins which include an EGFR binding armimmunoglobulin light chain (or V_(L)) variant comprising the amino acidsequence set forth in SEQ ID NO: 16 but having one or more of suchmutations and/or an immunoglobulin heavy chain (or V_(H)) variantcomprising the amino acid sequence set forth in SEQ ID NO: 2 but havingone or more of such mutations. In an embodiment of the invention, aCD28xEGFR antigen-binding protein includes an immunoglobulin light chainvariant comprising LCDR1, LCDR2 and LCDR3 wherein one or more (e.g., 1or 2 or 3) of such CDRs has one or more of such mutations (e.g.,conservative substitutions) and/or an immunoglobulin heavy chain variantcomprising HCDR1, HCDR2 and HCDR3 wherein one or more (e.g., 1 or 2 or3) of such CDRs has one or more of such mutations (e.g., conservativesubstitutions).

A “conservatively modified variant” or a “conservative substitution”,e.g., of an immunoglobulin chain set forth herein, refers to a variantwherein there is one or more substitutions of amino acids in apolypeptide with other amino acids having similar characteristics (e.g.charge, side-chain size, hydrophobicity/hydrophilicity, backboneconformation and rigidity, etc.). Such changes can frequently be madewithout significantly disrupting the biological activity of the antibodyor fragment. Those of skill in this art recognize that, in general,single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. (1987) Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4^(th) Ed.)). In addition,substitutions of structurally or functionally similar amino acids areless likely to significantly disrupt biological activity. The presentinvention includes EGFRxCD28 and anti-EGFR antigen-binding proteinsand/or binding arms comprising such conservatively modified variantimmunoglobulin chains.

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Alternatively, a conservativereplacement is any change having a positive value in the PAM250log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45.

Antigen-Binding Molecules

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-CD28 antibodies of the presentinvention can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity.

Use of the expression “anti-CD28 antibody” herein is intended to includeboth monospecific anti-CD28 antibodies as well as multispecific (e.g.,bispecific) antibodies comprising a CD28-binding arm and a second armthat binds a target antigen. Thus, the present invention includesbispecific antibodies wherein one arm of an immunoglobulin binds humanCD28, and the other arm of the immunoglobulin is specific for a targetantigen. The target antigen that the other arm of the CD28 bispecificantibody binds can be any antigen expressed on or in the vicinity of acell, tissue, organ, microorganism or virus, against which a targetedimmune response is desired. The CD28-binding arm can comprise any of theHCVR/LCVR or CDR amino acid sequences as set forth in Tables 3 and 8herein. In certain embodiments, the CD28-binding arm binds human CD28and induces human T-cell proliferation.

In the context of bispecific antibodies of the present invention whereinone arm of the antibody binds CD28 and the other arm binds a targetantigen, the target antigen can be a tumor-associated antigen, such asEGFR.

According to certain exemplary embodiments, the present inventionincludes bispecific antigen-binding molecules that specifically bindCD28 and EGFR. Such molecules may be referred to herein as, e.g.,“anti-CD28/anti-EGFR,” or “anti-CD28xEGFR,” or “CD28xEGFR” or“EGFRXCD28”, or “anti-EGFR/anti-CD28,” or “anti-EGFRxCD28,” or“EGFRxCD28” bispecific molecules, or “anti-EGFR X anti-CD28” or“anti-CD28 X anti-EGFR”, or other similar terminology.

According to certain exemplary embodiments, the bispecificantigen-binding molecules (e.g., bispecific antibody) may have aneffector arm and a targeting arm. The effector arm may be the firstantigen-binding domain (e.g., anti-CD28 antibody) that binds to theantigens on effector cells (e.g., T cells). The targeting arm may be thesecond antigen binding domain (e.g., anti-/EGFR antibody) that binds tothe antigens on target cells (e.g., tumor cells). According to certainexemplary embodiments, the effector arm binds to CD28 and the targetingarm binds to EGFR. The bispecific anti-CD28/EGFR may provideco-stimulatory signal to effector cells (e.g., T-cells).

As used herein, the expression “antigen-binding molecule” means aprotein, polypeptide or molecular complex comprising or consisting of atleast one complementarity determining region (CDR) that alone, or incombination with one or more additional CDRs and/or framework regions(FRs), specifically binds to a particular antigen. In certainembodiments, an antigen-binding molecule is an antibody or a fragment ofan antibody, as those terms are defined elsewhere herein.

As used herein, the expression “bispecific antigen-binding molecule”means a protein, polypeptide or molecular complex (e.g. an antibody orantigen-binding fragment thereof) comprising at least a firstantigen-binding domain and a second antigen-binding domain. Eachantigen-binding domain within the bispecific antigen-binding moleculecomprises at least one CDR that alone, or in combination with one ormore additional CDRs and/or FRs, specifically binds to a particularantigen. In the context of the present invention, the firstantigen-binding domain specifically binds a first antigen (e.g., CD28),and the second antigen-binding domain specifically binds a second,distinct antigen (e.g., EGFR).

In certain exemplary embodiments of the present invention, thebispecific antigen-binding molecule is a bispecific antibody. Eachantigen-binding domain of a bispecific antibody comprises a heavy chainvariable domain (HCVR) and a light chain variable domain (LCVR).

The first antigen-binding domain and the second antigen-binding domainmay be directly or indirectly connected to one another to form abispecific antigen-binding molecule of the present invention.Alternatively, the first antigen-binding domain and the second antigenbinding domain may each be connected to a separate multimerizing domain.The association of one multimerizing domain with another multimerizingdomain facilitates the association between the two antigen-bindingdomains, thereby forming a bispecific antigen-binding molecule. As usedherein, a “multimerizing domain” is any macromolecule, protein,polypeptide, peptide, or amino acid that has the ability to associatewith a second multimerizing domain of the same or similar structure orconstitution. For example, a multimerizing domain may be a polypeptidecomprising an immunoglobulin C_(H)3 domain. A non-limiting example of amultimerizing component is an Fc portion of an immunoglobulin(comprising a C_(H)2-C_(H)3 domain), e.g., an Fc domain of an IgGselected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as anyallotype within each isotype group.

Bispecific antigen-binding molecules of the present invention willtypically comprise two multimerizing domains, e.g., two Fc domains thatare each individually part of a separate antibody heavy chain. The firstand second multimerizing domains may be of the same IgG isotype such as,e.g., IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first andsecond multimerizing domains may be of different IgG isotypes such as,e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerizing domain is an Fc fragment or anamino acid sequence of 1 to about 200 amino acids in length containingat least one cysteine residues. In other embodiments, the multimerizingdomain is a cysteine residue, or a short cysteine containing peptide.Other multimerizing domains include peptides or polypeptides comprisingor consisting of a leucine zipper, a helix-loop motif, or a coiled-coilmotif.

Any bispecific antibody format or technology may be used to make thebispecific antigen-binding molecules of the present invention. Forexample, an antibody or antigen-binding fragment thereof having a firstantigen binding specificity can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody orantibody fragment having a second antigen-binding specificity to producea bispecific antigen-binding molecule. Specific exemplary bispecificformats that can be used in the context of the present inventioninclude, without limitation, e.g., scFv-based or diabody bispecificformats, IgG-scFv fusions, dual variable domain (OVO)-Ig, Quadroma,knobs-into-holes, common light chain (e.g., common light chain withknobs-intoholes, etc.), CrossMab, CrossFab, (SEEO)body, leucine zipper,Ouobody, IgG1/IgG2, dual acting Fab (OAF)-IgG, and Mab² bispecificformats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and referencescited therein, for a review of the foregoing formats).

In the context of bispecific antigen-binding molecules of the presentinvention, the multimerizing domains, e.g., Fc domains, may comprise oneor more amino acid changes (e.g., insertions, deletions orsubstitutions) as compared to the wild-type, naturally occurring versionof the Fc domain. For example, the invention includes bispecificantigen-binding molecules comprising one or more modifications in the Fcdomain that results in a modified Fc domain having a modified bindinginteraction (e.g., enhanced or diminished) between Fc and FcRn. In oneembodiment, the bispecific antigen-binding molecule comprises amodification in a C_(H)2 or a C_(H)3 region, wherein the modificationincreases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., LN/FIW or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/EID orT); or a modification at position 428 and/or 433 (e.g., UR/S/P/Q or K)and/or 434 (e.g., H/F or V); or a modification at position 250 and/or428; or a modification at position 307 or 308 (e.g., 308F, V308F), and434. In one embodiment, the modification comprises a 428L (e.g., M428L)and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g.,434Y) modification; a 252,254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 2500 and 428L modification (e.g., T250Q and M428L); anda 307 and/or 308 modification (e.g., 308F or 308P).

The present invention also includes bispecific antigen-binding moleculescomprising a first C_(H)3 domain and a second Ig C_(H)3 domain, whereinthe first and second Ig C_(H)3 domains differ from one another by atleast one amino acid, and wherein at least one amino acid differencereduces binding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference. In oneembodiment, the first Ig C_(H)3 domain binds Protein A and the second IgC_(H)3 domain contains a mutation that reduces or abolishes Protein Abinding such as an H95R modification (by IMGT exon numbering; H435R byEU numbering). The second C_(H)3 may further comprise a Y96Fmodification (by IMGT; Y436F by EU). Further modifications that may befound within the second C_(H)3 include: D16E, L 18M, N44S, K52N, V57M,and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V4221 by EU)in the case of IgG1 antibodies; N44S, K52N, and V821 (IMGT; N384S,K392N, and V4221 by EU) in the case of IgG2 antibodies; and Q15R, N44S,K52N, V57M, R69K, E79Q, and V821 (by IMGT; Q355R, N384S, K392N, V397M,R409K, E419Q, and V4221 by EU) in the case of IgG4 antibodies.

In certain embodiments, the Fc domain may be chimeric, combining Fcsequences derived from more than one immunoglobulin isotype. Forexample, a chimeric Fc domain can comprise part or all of a C_(H)2sequence derived from a human IgG1, human IgG2 or human IgG4 C_(H)2region, and part or all of a C_(H)3 sequence derived from a human IgG1,human IgG2 or human IgG4. A chimeric Fc domain can also contain achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” sequence, derived from a human IgG1, a human IgG2 or ahuman IgG4 hinge region, combined with a “lower hinge” sequence, derivedfrom a human IgG1, a human IgG2 or a human IgG4 hinge region. Aparticular example of a chimeric Fc domain that can be included in anyof the antigen-binding molecules set forth herein comprises, from N- toC-terminus: [IgG4 C_(H)1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4C_(H)2]-[IgG4 C_(H)3]. Another example of a chimeric Fc domain that canbe included in any of the antigen-binding molecules set forth hereincomprises, from N- to C-terminus: [IgG1 C_(H)1]-[IgG1 upper hinge]-[IgG2lower hinge]-[IgG4 C_(H)2]-[IgG1 C_(H)3]. These and other examples ofchimeric Fc domains that can be included in any of the antigen-bindingmolecules of the present invention are described in WO2014/022540 A1,Chimeric Fc domains having these general structural arrangements, andvariants thereof, can have altered Fc receptor binding, which in turnaffects Fc effector function.

Antibodies and antigen-binding fragments of the present inventioncomprise immunoglobulin chains including the amino acid sequencesspecifically set forth herein (and variants thereof) as well as cellularand in vitro post-translational modifications to the antibody orfragment. For example, the present invention includes antibodies andantigen-binding fragments thereof that specifically bind to EGFR andCD28 comprising heavy and/or light chain amino acid sequences set forthherein as well as antibodies and fragments wherein one or moreasparagine, serine and/or threonine residues is glycosylated, one ormore asparagine residues is deamidated, one or more residues (e.g., Met,Trp and/or His) is oxidized, the N-terminal glutamine is pyroglutamate(pyroE) and/or the C-terminal lysine or other amino acid is missing.

Epidermal Growth Factor Receptor (EGFR) Binding Molecules and Anti-EGFRAntigen-Binding Arms

The present invention includes multispecific (e.g., bispecific)antigen-binding proteins, e.g., antibodies or antigen-binding fragments,including one or more EGFR binding arms as well as one or more CD28binding arms.

An EGFR binding arm is the portion of a multispecific antigen-bindingprotein that confers EGFR binding upon the protein. In an embodiment ofthe invention, the EGFR binding arm of the bispecific antibodiesREGN7075, REGN6321, REGN6322 or REGN6323 blocks EGF binding to EGFR andin another embodiment of the invention, the EGFR binding arm does notblock EGF binding to EGFR. For example, an EGFR-binding arm of aY-shaped IgG antibody refers to a structural portion of the antibodythat confers binding specificity to the EGFR. For example, in anembodiment of the invention, an EGFR Binding Arm comprises HCDR1, LCDR1,HCDR2, LCDR2, HCDR3 and LCDR3; a HCVR (V_(H)) and an LCVR (V_(L)) and/ora HC and LC that binds specifically to EGFR.

In an embodiment of the invention, an EGFR binding arm includes a heavychain immunoglobulin that comprises a V_(H) including the combination ofheavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the corresponding lightchain immunoglobulin that comprises a V_(L) including the combination oflight chain CDRs (LCDR1, LCDR2 and LCDR3) which are set forth herein orin International patent application publication no. WO2014/004427.

“085N”; “086N”; “089N”; “102N”; “103N”; “116N”; “134P”; “136P”; “141P”;“142P”; “143P”; “144P”; “145P”; “147P”; “151P”; “153P”; “155P”; “157P”;“158P”; “159P”; “161P”; “163P”; “169P” and “171P” refers to theidentification number of several anti-EGFR monospecific antibodiesdescribed in WO2014/004427. The HCVR arm of these antibodies may be usedto combine with an anti-CD28 arm having HCVR amino acid sequences asdescribed in Table 3 and a common light chain with the amino acidsequences as described in Table 6 to prepare a bispecific antibody totarget a tumor cell expressing EGFR and a T cell expressing CD28.“REGN7075”, “REGN6321”, “REGN6322” or “REGN6323” also referred torespectively as “bsAb7075”, “bsAb6321”, “bsAb6322” or “bsAb6323” refersto bispecific antibodies comprising an EGFR Binding Arm including theimmunoglobulin heavy chain variable region (V_(H)) or full length heavychain (or a variant thereof) as set forth herein in Tables 1, 6 and 8and the corresponding immunoglobulin light chain variable region (V_(L))or full length light chain (or a variant thereof) set forth herein inTables 1, 6 and 8 or that comprise a V_(H) that comprises the CDRsthereof (HCDR1 (or a variant thereof), HCDR2 (or a variant thereof) andHCDR3 (or a variant thereof)) and the corresponding V_(L) that comprisesthe CDRs thereof (LCDR1 (or a variant thereof), LCDR2 (or a variantthereof) and LCDR3 (or a variant thereof)), e.g., wherein the variableregions and/or CDRs comprise the specific amino acid sequences describedherein and are not variants. Such a V_(H) may comprise variant aminoacid sequences wherein the CDR-Hs are not variants; and/or such a V_(L)may comprise variant amino acid sequences wherein the CDR-Ls are notvariants. Such EGFR binding arms may be referred to in the context of anEGFRxCD28 multispecific antigen-binding protein, e.g., 085Nx14226P2. Inan embodiment of the invention, the V_(H) is linked to an IgG constantheavy chain domain (e.g., IgG1 or IgG4 (e.g., comprising a S228Pmutation)) and/or the V_(L) is linked to a lambda or kappa constantlight chain domain.

The present invention also provides antigen-binding proteins, such asantibodies (e.g., human antibodies, monoclonal antibodies andrecombinant antibodies) and antigen-binding fragments thereof, thatspecifically bind to EGFR protein or an antigenic fragment thereof(e.g., the extracellular domain of EGFR). Antigen-binding proteins thatbind to the same epitope on EGFR as, or compete for binding to EGFR withany of the antigen-binding proteins set forth herein, are also part ofthe present invention.

Anti-EGFR antibodies and antigen-binding fragments thereof of thepresent invention include those comprising the CDRs (HCDR1, HCDR2 andHCDR3), V_(H) or full length immunoglobulin sequence of the 085N; 086N;089N; 102N; 103N; 116N; 134P; 136P; 141P; 142P; 143P; 144P; 145P; 147P;151P; 153P; 155P; 157P; 158P; 159P; 161P; 163P; 169P; 171P; mAb12999P2,mAb13008P2, mAb35193P2, mAb13006P2, REGN7075, REGN6321, REGN6322 orREGN6323 EGFR binding arm as set forth herein or in WO2014/004427 (or avariant thereof); and/or the CDRs (LCDR1, LCDR2 and LCDR3), V_(L) orfull length immunoglobulin sequence of the 085N; 086N; 089N; 102N; 103N;116N; 134P; 136P; 141P; 142P; 143P; 144P; 145P; 147P; 151P; 153P; 155P;157P; 158P; 159P; 161P; 163P; 169P; 171P; mAb12999P2, mAb13008P2,mAb35193P2, mAb13006P2, REGN7075, REGN6321, REGN6322 or REGN6323 EGFRbinding arm as set forth herein or in WO2014/004427 (or a variantthereof).

“REGN7075”, “REGN6321”, “REGN6322” or “REGN6323” may also refer toanti-EGFR antibodies and antigen-binding fragments thereof (e.g.,monospecific antibodies and fragments) including the immunoglobulinheavy chain variable region (V_(H)) or full length heavy chain set forthherein (or a variant thereof); and the corresponding immunoglobulinlight chain variable region (V_(L)) or full length light chain set forthherein (or a variant thereof); or that comprise a V_(H) that comprisesthe CDRs thereof (HCDR1 (or a variant thereof), HCDR2 (or a variantthereof) and HCDR3 (or a variant thereof)) and the corresponding V_(L)that comprises the CDRs thereof (LCDR1 (or a variant thereof), LCDR2 (ora variant thereof) and LCDR3 (or a variant thereof)), e.g., wherein thevariable regions and/or CDRs comprise the specific amino acid sequencesdescribed herein and are not variants. Such a V_(H) may comprise variantamino acid sequences wherein the CDR-Hs are not variants; and/or such aV_(L) may comprise variant amino acid sequences wherein the CDR-Ls arenot variants. In an embodiment of the invention, the V_(H) is linked toan IgG constant heavy chain domain (e.g., IgG1 or IgG4) and/or the V_(L)is linked to a lambda or kappa constant light chain domain.

The antigen-binding proteins REGN7075, REGN6321, REGN6322 and REGN6323are bispecific anti-EGFR antigen-binding proteins (e.g., an antibody orantigen-binding fragment thereof) having one HCVR arm that binds to EGFR(and/or one or more different antigens or different EGFR epitopes) andhaving one HCVR arm that binds to CD28 on a T cell.

Amino acid sequences of the immunoglobulin chains of REGN7075, REGN6321,REGN6322 and REGN6323 are set forth below:

EGFR Components of REGN7075HCVR of the EGFR arm from parental EGFR mAb12999P2):(SEQ ID NO: 2; CDRs underscored)QVQLQESGPGLVKPSETLSLTCTVSGDSIITFYWSWIRQPPGRGLEWIGYIYYSGITNYNPSLKSRVTISVDTSKNQVSLKLSSVTAADTAVYYCARVSEDSYFHYGMDVWGQGTTVTVSS (SEQ ID NO: 4)CDR-H1: G D S I I T F Y (SEQ ID NO: 6) CDR-H2: I Y Y S G I T(SEQ ID NO: 8) CDR-H3: A R V S E D S Y F H Y G M D VFull length heavy chain of the EGFR arm of the bispecific antibody REGN7075(SEQ ID NO: 24)QVQLQESGPGLVKPSETLSLTCTVSGDSIITFYWSWIRQPPGRGLEWIGYIYYSGITNYNPSLKSRVTISVDTSKNQVSLKLSSVTAADTAVYYCARVSEDSYFHYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGKLCVR of the EGFR and CD28 parental antibodies and the bispecific antibodyREGN7075 (SEQ ID NO: 16; CDRs underscored)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 18)CDR-L1: QSVSSSY (SEQ ID NO: 20) CDR-L2: GAS (SEQ ID NO: 22)CDR-L3: QQYGSSPWTFull length light chain of both EGFR and CD28 parental antibodies and thebispecifics (SEQ ID NO: 28)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC EGFR Components of REGN6321HCVR of the EGFR arm from parental EGFR mAb13008P2(SEQ ID NO: 30; CDRs underscored)EVQLVESGGGLVRPGGSLRLSCTASGFTFSTFIMFWVRQAPGKGLEYVSSISSNGGTIYYADSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCTRGGDFWSGYYPFDYWGQGTLVTVSS(SEQ ID NO: 32) CDR-H1: G F T F S T F I (SEQ ID NO: 34)CDR-H2: I S S N G GT I (SEQ ID NO: 36)CDR-H3: T R G G D F W S G Y Y P F D YFull length heavy chain of the EGFR arm of the bispecific antibody REGN6321(SEQ ID NO: 38)EVQLVESGGGLVRPGGSLRLSCTASGFTFSTFIMFWVRQAPGKGLEYVSSISSNGGTIYYADSVKGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCTRGGDFWSGYYPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKLCVR of the EGFR and CD28 parental antibodies and the bispecific antibodyREGN6321 (SEQ ID NO: 16; CDRs underscored)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 18)CDR-L1: QSVSSSY (SEQ ID NO: 20) CDR-L2: GAS (SEQ ID NO: 22)CDR-L3: QQYGSSPWTFull length light chain of both EGFR and CD28 parental antibodies and thebispecifics (SEQ ID NO: 28)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC EGFR Components of REGN6322HCVR of the EGFR arm from parental EGFR mAb35193P2:(SEQ ID NO: 40; CDRs underscored)EVQLVESGGGLVKPGGSLRLSCTASGFSFRDAWMTWVRQVPGKGLEWVGRIRNKIDGGTTDYNTPVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDIWNYVLFYYYGLDVWGQGTTVTV SS(SEQ ID NO: 42) CDR-H1: G F S F R D A W (SEQ ID NO: 44)CDR-H2: I R N K I D G G T T (SEQ ID NO: 46)CDR-H3: T T D I W N Y V L F Y Y Y G L D VFull length heavy chain of the EGFR arm of the bispecific antibody REGN6322(SEQ ID NO: 48)EVQLVESGGGLVKPGGSLRLSCTASGFSFRDAWMTWVRQVPGKGLEWVGRIRNKIDGGTTDYNTPVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDIWNYVLFYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKLCVR of the EGFR and CD28 parental antibodies and the bispecific antibodyREGN6322 (SEQ ID NO: 16; CDRs underscored)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 18)CDR-L1: QSVSSSY (SEQ ID NO: 20) CDR-L2: GAS (SEQ ID NO: 22)CDR-L3: QQYGSSPWTFull length light chain of both EGFR and CD28 parental antibodies and thebispecifics (SEQ ID NO: 28)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC EGFR Components of REGN6323HCVR of the EGFR arm from parental EGFR mAb13006P2:(SEQ ID NO: 50; CDRs underscored)QVQLQESGPGLVKPSETLSLTCTVSDDSIISYYWSWIRQPPGKGLEWIGYIYYSGRTNYNPSLKSRVTISVDTSKNQVSLKLNSVIAADTAVYYCARVSEDSYYHYGMDVWGQGTTVTVSS(SEQ ID NO: 52) CDR-H1: D D S I I S Y Y (SEQ ID NO: 54)CDR-H2: I Y Y S G R T (SEQ ID NO: 56)CDR-H3: A R V S E D S Y Y H Y G M D VFull length heavy chain of the EGFR arm of the bispecific antibody REGN6323(SEQ ID NO: 58)QVQLQESGPGLVKPSETLSLTCTVSDDSIISYYWSWIRQPPGKGLEWIGYIYYSGRTNYNPSLKSRVTISVDTSKNQVSLKLNSVIAADTAVYYCARVSEDSYYHYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGKLCVR of the EGFR and CD28 parental antibodies and the bispecific antibodyREGN6323 (SEQ ID NO: 16; CDRs underscored)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 18)CDR-L1: QSVSSSY (SEQ ID NO: 20) CDR-L2: GAS (SEQ ID NO: 22)CDR-L3: QQYGSSPWTFull length light chain of both EGFR and CD28 parental antibodies and thebispecifics (SEQ ID NO: 28)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In an embodiment of the invention, an mAb12999P2, mAb13008P2,mAb35193P2, or mAb13006P2 heavy chain is paired with a light chaincomprising an amino acid sequence selected from:

(SEQ ID NO: 16) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG QGTKVEIK; and(SEQ ID NO: 67) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFG QGTRLEIK.

CD28 (Cluster of Differentiation 28) Binding Arms

The present invention includes multispecific (e.g., bispecific)antigen-binding proteins, e.g., antibodies or antigen-binding fragments,including one or more CD28 Binding Arms as well as one or more EGFRBinding Arms.

A CD28 Binding Arm is the portion of a multispecific antigen-bindingprotein that confers CD28 binding upon the protein. For example, aCD28-binding arm of a Y-shaped IgG antibody refers to a structuralportion of the antibody that confers binding specificity to the CD28.For example, in an embodiment of the invention, a CD28 Binding Armcomprises HCDR1, LCDR1, HCDR2, LCDR2, HCDR3 and LCDR3; a HVCR (V_(H))and an LCVR (V_(L)) and/or a HC and LC that binds specifically to CD28.

In an embodiment of the invention, a CD28 Binding Arm includes a heavychain immunoglobulin that comprises a V_(H) including the combination ofheavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the corresponding lightchain immunoglobulin that comprises a V_(L) including the combination oflight chain CDRs (LCDR1, LCDR2 and LCDR3) which are set forth herein. Inan embodiment of the invention, a CD28 Binding Arm includes a heavychain variable region (V_(H)) and the corresponding light chain variableregion (V_(L)) set forth herein.

Amino acid sequences of the immunoglobulin chains of parental anti-CD28antibodies14226P2, 14193P2 and 14216P2 are set forth below:

Parental CD28 mAbs used to prepare bispecifics mAb14226P2HCVR of mAb14226P2QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSS(SEQ ID NO: 10) CDR-H1: GGSISSYY (SEQ ID NO: 12)CDR-H2: IYYSGIT (SEQ ID NO: 6) CDR-H3: ARWGVRRDYYYYGMDVFull length heavy chain of mAb14226P2QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 14) (SEQ ID NO: 26) LCVR of mAb 14226P2EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 16)CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20)CDR-L3: QQYGSSPWT (SEQ ID NO: 22) Full length light chain of mAb14226P2EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28) mAb14193P2 HCVR of mAb14193P2QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYAGNNKYYADSVKGRFTVSRDNSKKTLYLQMNSLRSEDTAVYYCAKDSYYDFLTDPDVLDIWGQGTMVTVS S(SEQ ID NO: 59) CDR-H1: GFTFSSYG (SEQ ID NO: 60)CDR-H2: ISYAGNNK (SEQ ID NO: 61)CDR-H3: AKDSYYDFLTDPDVLDI (SEQ ID NO: 62) LCVR of mAb 14193P2EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 16)CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20)CDR-L3: QQYGSSPWT (SEQ ID NO: 22) mAb14216P2 HCVR of mAb 14216P2EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNNMHWVRQAPGKGLEYVSGISSNGGRTYYADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFCTRDDELLSFDYWGQGTLVTVSS(SEQ ID NO: 63) CDR-H1: GFTFSRNN (SEQ ID NO: 64)CDR-H2: ISSNGGRT (SEQ ID NO: 65) CDR-H3: TRDDELLSFDY (SEQ ID NO: 66)[0125] LCVR of mAb14216P2EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 16)CDR-L1: QSVSSSY (SEQ ID NO: 18) CDR-L2: GAS (SEQ ID NO: 20)CDR-L3: QQYGSSPWT (SEQ ID NO: 22)

In an embodiment of the invention, a 14226P2, 14193P2 or 14216P2 heavychain is paired with a light chain comprising an amino acid sequenceselected from:

(SEQ ID NO: 16) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG QGTKVEIK; and(SEQ ID NO: 67) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFG QGTRLEIK.

“14226P2”, “14193P2” or “14216P2” or “mAb14226P2”, “mAb14193P2” or“mAb14216P2” refer to the parental monospecific antibodies from whichthe CD28 arms of the bispecifics described herein are obtained. CD28Binding Arms including the immunoglobulin heavy chain variable region(V_(H)) or full length heavy chain set forth herein (or a variantthereof); and the corresponding immunoglobulin light chain variableregion (V_(L)) or full length light chain set forth herein (or a variantthereof); or that comprise a V_(H) that comprises the CDRs thereof(HCDR1 (or a variant thereof), HCDR2 (or a variant thereof) and HCDR3(or a variant thereof)) and the corresponding V_(L) that comprises theCDRs thereof (LCDR1 (or a variant thereof), LCDR2 (or a variant thereof)and LCDR3 (or a variant thereof)), e.g., wherein the variable regionsand/or CDRs comprise the specific amino acid sequences described hereinand which are not variants. Such a V_(H) may comprise variant amino acidsequences wherein the CDR-Hs are not variants; and/or such a V_(L) maycomprise variant amino acid sequences wherein the CDR-Ls are notvariants. Such CD28 binding arms may be referred to in the context of anEGFRxCD28 multispecific antigen-binding protein, e.g., 085Nx14226P2. Inan embodiment of the invention, the V_(H) is linked to an IgG constantheavy chain domain (e.g., IgG1 or IgG4) and/or the V_(L) is linked to alambda or kappa constant light chain domain.

The present invention also provides antigen-binding proteins, such asantibodies (e.g., human antibodies, monoclonal antibodies andrecombinant antibodies) and antigen-binding fragments thereof, thatspecifically bind to CD28 protein or an antigenic fragment thereof(e.g., the extracellular domain of CD28). Antigen-binding proteins thatbind to the same epitope on CD28 as, or compete for binding to CD28 withany of the antigen-binding proteins set forth herein, are also part ofthe present invention.

The multispecific EGFRxCD28 antigen-binding proteins of the presentinvention which bind to CD28 on the surface of a T-cell and agonize CD28signaling enhancing activation and/or proliferation of the T-cell may bereferred to, herein, as “co-stimulatory” or “costimulatory”. T-cellactivation is initiated upon binding of the T-cell Receptor (TCR)/CD3complex to peptide-MHC complexes (“signal 1”); activation is thenenhanced by engagement of a second “co-stimulatory” receptor, such asthe CD28 receptor on T-cells binding to its cognate ligand(s) on thetarget cell (“signal 2”). For example, activation of a T-cell by a CD28bi-specific antibody may be caused by amplification of signals inresponse to endogenous tumor antigen recognition by the TCR/CD3 complex,or in response to “signal 1” activation via a CD3-bispecific.

Multispecific Antigen-Binding Proteins

The present invention provides antigen-binding proteins which aremultispecific (e.g., bispecific) and bind at least to EGFR and CD28. Asused herein, such an antibody is referred to in the format AxB wherein Arefers to a binding arm in the multispecific molecule that binds to EGFRand B refers to a binding arm in the multispecific molecule that bindsto CD28, or vice versa. EGFRxCD28 or CD28xEGFR refer to a multispecificantigen binding protein that binds to EGFR and CD28. The specific EGFRand CD28 binding arms in a multispecific antigen-binding protein alsomay be specified in an AxB format wherein A refers to a specific arm andB refers to another specific arm. For example, 085Nx14226P2 refers to amultispecific antigen-binding protein that has the anti-EGFR bindingarms of 085N as set forth herein and the anti-CD28 binding arms of14426P2 as set forth herein. 085N, for example, is a binding armincluding the 085N immunoglobulin heavy and light chains or variableregions thereof or CDRs thereof whose sequences are specifically setforth herein or are variants thereof.

In certain embodiments, the multispecific antigen-binding proteinscomprise bispecific antigen-binding proteins. As used herein, theexpression “bispecific antigen-binding protein” means a protein,polypeptide or molecular complex (e.g., antibody or antigen-bindingfragment thereof) comprising at least a first antigen-binding domain anda second antigen-binding domain. Each antigen-binding domain within thebispecific antigen-binding molecule comprises at least one CDR thatalone, or in combination with one or more additional CDRs and/or FRs,specifically binds to a particular antigen. In the context of thepresent invention, the first antigen-binding domain specifically binds afirst antigen (e.g., CD28), and the second antigen-binding domainspecifically binds a second, distinct antigen (e.g., EGFR).

Multispecific binding refers to binding to two or more differentepitopes (EGFR and CD28 or more) which may be on the same or ondifferent antigens. Multispecific includes bispecific, trispecific andtetraspecific.

The present invention includes any of the following multispecificantigen-binding proteins (e.g., bispecific antibodies or antigen-bindingfragments thereof): REGN7075, REGN6321, REGN6322, REGN6323, as well asbispecific antibodies prepared by combining any of the EGFR HCVR arms ofTable 1 (e.g., HCVR arms of the parental monoclonal antibodiesmAb12999P2, mAb13008P2, mAb35193P2 and mAb13006P2) with any of the CD28HCVR arms of Table 3 (e.g. HCVR arms of the parental mAb14226, mAb14193and mAb14216) and methods of use thereof as set forth herein.

Polynucleotides and Methods of Making

An isolated polynucleotide encoding the immunoglobulin chains of anyEGFRxCD28 multispecific antigen-binding protein or anti-EGFRantigen-binding protein set forth herein forms part of the presentinvention as does a vector comprising the polynucleotide and/or a hostcell (e.g., Chinese hamster ovary (CHO) cell) comprising thepolynucleotide, vector or antigen-binding protein set forth herein.

A polynucleotide includes DNA and RNA. The present invention includesany polynucleotide of the present invention, for example, encoding animmunoglobulin V_(H), V_(L), CDR-H, CDR-L, HC or LC of an EGFR BindingArm and/or a CD28 Binding Arm, optionally, which is operably linked to apromoter or other expression control sequence. For example, the presentinvention provides any polynucleotide (e.g., DNA) that includes anucleotide sequence set forth in Table 2 and a nucleotide sequence setforth in Table 4.

The present invention includes a polynucleotide comprising a nucleotidesequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 15, 17, 19, 21,29, 31, 33, 35, 39, 41, 43, 45, 49, 51, 53 and/or 55, optionallyoperably linked to a promoter or other expression control sequence orother polynucleotide sequence.

In general, a “promoter” or “promoter sequence” is a DNA regulatoryregion capable of binding an RNA polymerase in a cell (e.g., directly orthrough other promoter-bound proteins or substances) and initiatingtranscription of a coding sequence. A promoter may be operably linked toother expression control sequences, including enhancer and repressorsequences and/or with a polynucleotide of the invention. Promoters whichmay be used to control gene expression include, but are not limited to,cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062),the SV40 early promoter region (Benoist, et al., (1981) Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., (1980) Cell 22:787-797), theherpes thymidine kinase promoter (Wagner, et al., (1981) Proc. Natl.Acad. Sci. USA 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster, et al., (1982) Nature 296:39-42);prokaryotic expression vectors such as the beta-lactamase promoter(VIlla-Komaroff, et al., (1978) Proc. Natl. Acad. Sci. USA75:3727-3731), or the tac promoter (DeBoer, et al., (1983) Proc. Natl.Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American (1980) 242:74-94; and promoter elementsfrom yeast or other fungi such as the Ga/4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or thealkaline phosphatase promoter.

A polynucleotide encoding a polypeptide is “operably linked” to apromoter or other expression control sequence when, in a cell or otherexpression system, the sequence directs RNA polymerase mediatedtranscription of the coding sequence into RNA, preferably mRNA, whichthen may be RNA spliced (if it contains introns) and, optionally,translated into a protein encoded by the coding sequence.

The present invention includes polynucleotides encoding immunoglobulinpolypeptide chains which are variants of those whose nucleotide sequenceis specifically set forth herein. A “variant” of a polynucleotide refersto a polynucleotide comprising a nucleotide sequence that is at leastabout 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%)identical to a referenced nucleotide sequence that is set forth herein;when the comparison is performed by a BLAST algorithm wherein theparameters of the algorithm are selected to give the largest matchbetween the respective sequences over the entire length of therespective reference sequences (e.g., expect threshold: 10; word size:28; max matches in a query range: 0; match/mismatch scores: 1, −2; gapcosts: linear). In an embodiment of the invention, a variant of anucleotide sequence specifically set forth herein comprises one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) point mutations,insertions (e.g., in frame insertions) or deletions (e.g., in framedeletions) of one or more nucleotides. Such mutations may, in anembodiment of the invention, be missense or nonsense mutations. In anembodiment of the invention, such a variant polynucleotide encodes animmunoglobulin polypeptide chain which can be incorporated into an EGFRBinding Arm and/or CD28 Binding Arm, i.e., such that the protein retainsspecific binding to EGFR and/or CD28.

Eukaryotic and prokaryotic host cells, including mammalian cells, may beused as hosts for expression of an anti-EGFR and EGFRxCD28antigen-binding protein (e.g., antibody or antigen-binding fragmentthereof) or an antigen binding arm thereof. Such host cells are wellknown in the art and many are available from the American Type CultureCollection (ATCC). These host cells include, inter alia, Chinese hamsterovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number ofother cell lines. Mammalian host cells include human, mouse, rat, dog,monkey, pig, goat, bovine, horse and hamster cells. Other cell linesthat may be used are insect cell lines (e.g., Spodoptera frugiperda orTrichoplusia nl), amphibian cells, bacterial cells, plant cells andfungal cells. Fungal cells include yeast and filamentous fungus cellsincluding, for example, Pichia, Pichia pastoris, Pichia finlandica,Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichiaminuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichiathermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi,Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Physcomitrella patens and Neurospora crassa. The present inventionincludes an isolated host cell (e.g., a CHO cell or any type of hostcell set forth above) comprising an anti-EGFR antibody (e.g., an EGFRantibody found in WO2014/004427) or an anti-EGFR x anti-CD28antigen-binding protein of the present invention, such as REGN7075;REGN6321; REGN6322; REGN6323 and the anti-EGFR X anti-CD28antigen-binding proteins shown in Tables 9A, 9B or 9C, or apolynucleotide encoding an immunoglobulin (Ig) heavy and/or light chainthereof); and/or one or more polynucleotides encoding the EGFR bindingarm and CD28 binding arm of a multispecific antigen-binding protein ofthe present invention.

The present invention also includes a cell which is expressing an EGFRand/or CD28 or an antigenic fragment or fusion thereof (e.g., His₆ (SEQID NO: 68), Fc and/or myc) which is bound by an EGFRxCD28 and/oranti-EGFR antigen-binding protein of the present invention (e.g., anantibody or antigen-binding fragment thereof), for example, REGN7075,REGN6321, REGN6322, REGN6323, as well as bispecific antibodies preparedby combining any of the EGFR HCVR arms of Table 1 (e.g., HCVR arms ofthe parental monoclonal antibodies mAb12999P2, mAb13008P2, mAb35193P2and mAb13006P2) with any of the CD28 HCVR arms of Table 3 (e.g. HCVRarms of the parental mAb14226, mAb14193 and mAb14216), or any of thebispecific antibodies shown in Tables 9A, 9B and 9C, for example,wherein the cell is in the body of a subject or is in vitro.

In addition, the present invention also provides a complex comprising anEGFRxCD28, and/or anti-EGFR antigen-binding protein of the presentinvention, e.g., antibody or antigen-binding fragment thereof, asdiscussed herein complexed with EGFR and/or CD28 polypeptide or anantigenic fragment thereof or fusion thereof and/or with a secondaryantibody or antigen-binding fragment thereof (e.g., detectably labeledsecondary antibody) that binds specifically to the anti-EGFR orEGFRxCD28 antibody or fragment. In an embodiment of the invention, thecomplex is in vitro (e.g., is immobilized to a solid substrate) or is inthe body of a subject. In an embodiment of the invention, the EGFR is onthe surface of a tumor cell and the CD28 is on the surface of an immunecell, e.g., a T-cell. In an embodiment of the invention, the T-cell isactivated.

There are several methods by which to produce recombinant antibodieswhich are known in the art. One example of a method for recombinantproduction of antibodies is disclosed in U.S. Pat. No. 4,816,567.Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,biolistic injection and direct microinjection of the DNA into nuclei. Inaddition, nucleic acid molecules may be introduced into mammalian cellsby viral vectors. Methods of transforming cells are well known in theart. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461and 4,959,455.

The present invention includes recombinant methods for making ananti-EGFR x anti-CD28 (e.g., REGN7075, REGN6321, REGN6322 or REGN6323)antigen-binding protein of the present invention, such as an antibody orantigen-binding fragment thereof of the present invention, or animmunoglobulin chain thereof, comprising

-   -   (i) introducing, into a host cell, one or more polynucleotides        encoding the light and heavy immunoglobulin chains encoding the        EGFRxCD28 or anti-EGFR antigen-binding protein's antigen binding        arms for example, wherein the polynucleotide is in a vector;        and/or integrates into the host cell chromosome and/or is        operably linked to a promoter;    -   (ii) culturing the host cell (e.g., CHO or Pichia or Pichia        pastoris) under conditions favorable to expression of the        polynucleotide and,    -   (iii) optionally, isolating the antigen-binding protein (e.g.,        antibody or antigen-binding fragment) or chain from the host        cell and/or medium in which the host cell is grown. The present        invention also includes anti-EGFR or EGFRxCD28 antigen-binding        proteins, such as antibodies and antigen-binding fragments        thereof, which are the product of the production methods set        forth herein, and, optionally, the purification methods set        forth herein.

In an embodiment of the invention, a method for making an EGFRxCD28(e.g., REGN7075, REGN6321, REGN6322 or REGN6323) antigen-bindingprotein, e.g., antibody or antigen-binding fragment thereof, includes amethod of purifying the antigen-binding protein, e.g., by columnchromatography, precipitation and/or filtration. As discussed, theproduct of such a method also forms part of the present invention.

Sequence Variants

The antibodies and bispecific antigen-binding molecules of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the individual antigen-binding domains werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germ line sequences availablefrom, for example, public antibody sequence databases. Theantigen-binding molecules of the present invention may compriseantigen-binding fragments which are derived from any of the exemplaryamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antigen-binding domain wasoriginally derived. In other embodiments, only certain residues aremutated back to the original germline sequence, e.g., only the mutatedresidues found within the first 8 amino acids of FR1 or within the last8 amino acids of FR4, or only the mutated residues found within CDR1,CDR2 or CDR3. In other embodiments, one or more of the framework and/orCDR residue(s) are mutated to the corresponding residue(s) of adifferent germline sequence (i.e., a germline sequence that is differentfrom the germ line sequence from which the antigen-binding domain wasoriginally derived). Furthermore, the antigen-binding domains maycontain any combination of two or more germline mutations within theframework and/or CDR regions, e.g., wherein certain individual residuesare mutated to the corresponding residue of a particular germ linesequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residueof a different germline sequence. Once obtained, antigen-binding domainsthat contain one or more germline mutations can be easily tested for oneor more desired properties such as improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Bispecific antigen-binding molecules comprising oneor more antigen-binding domains obtained in this general manner areencompassed within the present invention.

The present invention also includes antigen-binding molecules whereinone or both antigen-binding domains comprise variants of any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein having oneor more conservative substitutions. For example, the present inventionincludes antigen-binding molecules comprising an antigen-binding domainhaving HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 orfewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein. A “conservative amino acid substitution” isone in which an amino acid residue is substituted by another amino acidresidue having a side chain (R group) with similar chemical properties(e.g., charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. Examples of groups of amino acids that have side chains withsimilar chemical properties include (1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl sidechains: serine and threonine; (3) amide-containing side chains:asparagine and glutamine; (4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, andhistidine; (6) acidic side chains: aspartate and glutamate, and (7)sulfur-containing side chains are cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet aL. (1992) Science 256: 1443-1445. A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

The present invention also includes antigen-binding molecules comprisingan antigen binding domain with an HCVR, LCVR, and/or CDR amino acidsequence that is substantially identical to any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein. The term “substantialidentity” or “substantially identical,” when referring to an amino acidsequence means that two amino acid sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 95% sequence identity, even more preferably at least 98% or 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. In cases where two ormore amino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et aL. (1990) J. Mol. Biol. 215:403-410 and Altschul et aL.(1997) Nucleic Acids Res. 25:3389-402.

Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-EGFR Xanti-CD28 bispecific antigen binding molecules are provided comprisingan Fc domain comprising one or more mutations which enhance or diminishantibody binding to the FcRn receptor, e.g., at acidic pH as compared toneutral pH. For example, the present invention includes antibodies andantigen binding molecules comprising a mutation in the C_(H)2 or aC_(H)3 region of the Fc domain, wherein the mutation(s) increases theaffinity of the Fc domain to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0). Such mutationsmay result in an increase in serum half-life of the antibody whenadministered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position

-   -   250 (e.g., E or Q);    -   250 and 428 (e.g., L or F);    -   252 (e.g., L/Y/F/W or T),    -   254 (e.g., S or T), and/or    -   256 (e.g., S/R/Q/E/D or T);        or a modification at position    -   428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or    -   434 (e.g., H/F or Y);        or a modification at position    -   250 and/or 428;        or a modification at position    -   307 or 308 (e.g., 308F, V308F), and/or    -   434.

In one embodiment, the modification comprises a

-   -   428L (e.g., M428L) and 434S (e.g., N434S) modification;    -   a 428L, 2591 (e.g., V2591), and 308F (e.g., V308F) modification;    -   a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;    -   a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification;    -   a 2500 and 428L modification (e.g., T250Q and M428L); and/or    -   a 307 and/or 308 modification (e.g., 308F or 308P).

For example, the present invention includes EGFRxCD28 bispecific antigenbinding molecules comprising an Fc domain comprising one or more pairsor groups of mutations selected from the group consisting of:

-   -   2500 and 248L (e.g., T250Q and M248L);    -   252Y, 254T and 256E (e.g., M252Y, S254T and T256E);    -   428L and 434S (e.g., M428L and N434S); and    -   433K and 434F (e.g., H433K and N434F).

All possible combinations of the foregoing Fc domain mutations, andother mutations within the antibody variable domains disclosed herein,are contemplated within the scope of the present invention.

Biological Characteristics of the Antibodies and Antigen-BindingMolecules

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human CD28 and EGFR with high affinity. The presentinvention also includes antibodies and antigen binding fragments thereofthat bind human CD28 and/or EGFR with medium or low affinity, dependingon the therapeutic context and particular targeting properties that aredesired. For example, in the context of a bispecific antigen-bindingmolecule, wherein one arm binds CD28 and another arm binds a targetantigen (e.g., EGFR), it may be desirable for the target antigen-bindingarm to bind the target antigen with high affinity while the anti-CD28arm binds CD28 with only moderate or low affinity. In this manner,preferential targeting of the antigen-binding molecule to cellsexpressing the target antigen may be achieved while avoidinggeneral/untargeted CD28 binding and the consequent adverse side effectsassociated therewith.

According to certain embodiments, the present invention includesantibodies and antigen-binding fragments of antibodies that bind humanCD28 (e.g., at 25° C.) with a K_(D) of less than about 200 nM asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Examples 1 and 13 herein. In certain embodiments, theantibodies or antigen-binding fragments of the present invention bindCD28 with a K_(D) of less than about 100 nM, less than about 90 nM, lessthan about 80 nM, less than about 60 nM, less than about 40 nM, lessthan about 30 nM, less than 20 nM, less than 10 nM, or less than 5 nM asmeasured by surface plasmon resonance, e.g., using an assay format asdefined in Examples 1 and 13 herein, or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention bind CD28 with a K_(D) between from about 5 nM toabout 20 nM.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind CD28 with a dissociative half-life (t½) ofgreater than about 3 minutes as measured by surface plasmon resonance at25° C. or 37° C., e.g., using an assay format as defined in Examples 1and 13 herein, or a substantially similar assay. In certain embodiments,the antibodies or antigen-binding fragments of the present inventionbind CD28 with a t½ of greater than about 5 minutes, greater than about10 minutes, greater than about 20 minutes, greater than about 30minutes, greater than about 40 minutes, greater than about 50 minutes,greater than about 60 minutes, greater than about 70 minutes, greaterthan about 80 minutes, greater than about 90 minutes, greater than about100 minutes, greater than about 200 minutes, greater than about 300minutes, greater than about 400 minutes, greater than about 500 minutes,greater than about 600 minutes, greater than about 700 minutes, greaterthan about 800 minutes, greater than about 900 minutes, greater thanabout 1000 minutes, or greater than about 1200 minutes, as measured bysurface plasmon resonance at 25° C. or 37° C., e.g., using an assayformat as defined in Examples 1 and 13 herein, or a substantiallysimilar assay.

The present invention includes bispecific antigen-binding molecules(e.g., bispecific antibodies) which are capable of simultaneouslybinding to human CD28 and human EGFR. According to certain embodiments,the bispecific antigen-binding molecules of the invention specificallyinteract with cells that express CD28 and/or EGFR. The extent to which abispecific antigen-binding molecule binds cells that express CD28 and/orEGFR can be assessed by fluorescence activated cell sorting (FACS), asillustrated in Examples 10A and 10B herein. For example, the presentinvention includes bispecific antigen-binding molecules whichspecifically bind human cell lines which express CD28 but not EGFR(e.g., Jurkat cell), and human ovarian cancer cell lines which expressEGFR but not CD28 (e.g., PEO1). In some embodiments, the bispecificantigen-binding molecules bind to CD28-expressing human or cynomolgusT-cells with an EC50 value less than 1×10⁻⁵ M. In some embodiments, thebispecific antigen-binding molecules bind to CD28-expressing human orcynomolgus T-cells with an EC50 value of between 1×10⁻¹² M and 1×10⁻⁵ M.In certain embodiments, the bispecific antigen-binding molecules bind toCD28-expressing human or cynomolgus T-cells with an EC50 value ofbetween 1×10⁻⁹ M and 1×10⁻⁵ M. In certain embodiments, the bispecificantigen binding molecules bind to the surface of cell lines expressingEGFR with an EC₅₀ of less than about 2.5×10⁻⁸ M. The binding of thebispecific antigen binding molecules to the surface of cells or celllines can be measured by an in vitro FACS binding assay as described inExamples 10A and 10B.

EGFRxCD28 antigen-binding proteins set forth herein, e.g., comprisingvariant immunoglobulin chains, may exhibit one or more of the followingproperties:

-   -   Reduces the growth and/or survival of human tumor cells (e.g.,        A431 tumor cells) in a mouse (e.g., VH mouse (Rag2^(null)/γ_(c)        ^(null)/huSirp-a/huTPO)) engrafted with CD34⁺ cells (e.g., fetal        liver CD34⁺ cells); optionally wherein the EGFRxCD28 is        administered in association with a PD1 antagonist (e.g.,        anti-PD1 such as cemiplimab);    -   Reduces the growth and/or survival of human tumor cells (e.g.,        A431 tumor cells) in a mouse (e.g., (NOD/SCID/γ_(c) ^(null)))        comprising human PBMCs (e.g., wherein human PBMCs mixed with the        tumor cells are implanted subcutaneously into the mouse);        optionally wherein the EGFRxCD28 is administered in association        with a PD1 antagonist (e.g., anti-PD1 such as cemiplimab);    -   Reduces the growth and/or survival of human tumor cells (e.g.,        A549 tumor cells) in a mouse (e.g., NOD/SCID/γ_(c) ^(null))        comprising human PBMCs (e.g., wherein human PBMCs are implanted        intraperitoneally and the tumor cells are implanted        subcutaneously into the mouse); optionally wherein the EGFRxCD28        is administered in association with a PD1 antagonist (e.g.,        anti-PD1 such as cemiplimab);    -   Binds to Jurkat CD28⁺ cells;    -   Binds to PE01 EGFR+ cells; and/or    -   Does not cause a significant cytokine release (e.g.,        interferon-gamma, IL-2, IL-6, IL-8 and/or IL-10) when        administered to cynomolgus monkeys, e.g., at 10 mg/kg.

The present invention includes anti-EGFR and EGFRxCD28 bispecificantigen-binding molecules which are capable of depleting tumor cells ina subject (see, e.g., Examples 3-5). For example, according to certainembodiments, anti-EGFR and EGFRxCD28 bispecific antigen-bindingmolecules are provided, wherein a single administration of theantigen-binding molecule to a subject at a therapeutically effectivedose causes a reduction in the number of tumor cells in the subject.

The present invention includes anti-EGFR X anti-CD28 bispecificantigen-binding molecules which are capable of binding to a variety oftumor cells, including A375 melanoma cells, 22RV1 prostate cells, PEO1ovarian cells, CAPAN2 pancreatic cells, SW1990 pancreatic cells and H292lung cells (See Examples 10A and 10B). As such, the bispecificantibodies of the invention may prove useful in treating a multitude ofcancer indications.

The present invention includes anti-EGFR X anti-CD28 bispecificantigen-binding molecules which are capable of enhancing the cytotoxicpotency of anti-tumor specific antigen (TSA) X anti-CD3 bispecificantibodies across a variety of cell lines (See Example 11). Using thisapproach, the TSA X CD3 bispecific antibody may include anti-STEAP2 Xanti-CD3, anti-PSMA X anti-CD3, or anti-MUC16 X anti-CD3 bispecifics.The anti-EGFR X anti-CD28 antibodies may also prove useful when combinedwith a checkpoint inhibitor, for example, an antibody to PD-1 or PD-L1,or any other checkpoint inhibitor (See Example 9). In certainembodiments, it may be beneficial to combine the anti-EGFR X anti-CD28with both an anti-TSA X anti-CD3 bispecific plus a checkpoint inhibitor.

The antibodies of the invention were also shown to bind certain epitopeson human EGFR using hydrogen-deuterium exchange (See Example 12). Inparticular, certain of the antibodies of the invention were found tobind to/interact with amino acid residues 345-368 of human EGFR (SEQ IDNO: 70), amino acid residues 399-416 of human EGFR (SEQ ID NO: 71) andamino acid residues 133-154 of human EGFR (SEQ ID NO: 72).

Epitope Mapping and Related Technologies

The epitope on CD28 or EGFR to which the antigen-binding molecules ofthe present invention bind may consist of a single contiguous sequenceof 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) amino acids of a CD28 protein or a EGFR protein.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) of CD28 or EGFR. The antibodies ofthe invention may interact with amino acids contained within a CD28monomer, or may interact with amino acids on two different CD28 chainsof a CD28 dimer. The term “epitope,” as used herein, refers to anantigenic determinant that interacts with a specific antigen bindingsite in the variable region of an antibody molecule known as a paratope.A single antigen may have more than one epitope. Thus, differentantibodies may bind to different areas on an antigen and may havedifferent biological effects. Epitopes may be either conformational orlinear. A conformational epitope is produced by spatially juxtaposedamino acids from different segments of the linear polypeptide chain. Alinear epitope is one produced by adjacent amino acid residues in apolypeptide chain. In certain circumstance, an epitope may includemoieties of saccharides, phosphoryl groups, or sulfonyl groups on theantigen.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antigen-binding domain of an antibody“interacts with one or more amino acids” within a polypeptide orprotein. Exemplary techniques that can be used to determine an epitopeor binding domain of a particular antibody or antigen-binding domaininclude, e.g., routine crossblocking assay such as that described inAntibodies, Harlow and Lane (Cold Spring Harbor Press, Cold SpringHarb., NY), point mutagenesis (e.g., alanine scanning mutagenesis,arginine scanning mutagenesis, etc.), peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), protease protection, and peptidecleavage analysis. In addition, methods such as epitope excision,epitope extraction and chemical modification of antigens can be employed(Tomer, 2000, Protein Science 9:487-496). Another method that can beused to identify the amino acids within a polypeptide with which anantibody interacts is hydrogen/deuterium exchange detected by massspectrometry. In general terms, the hydrogen/deuterium exchange methodinvolves deuterium-labeling the protein of interest, followed by bindingthe antibody to the deuterium-labeled protein. Next, theprotein/antibody complex is transferred to water to allowhydrogen-deuterium exchange to occur at all residues except for theresidues protected by the antibody (which remain deuterium-labeled).After dissociation of the antibody, the target protein is subjected toprotease cleavage and mass spectrometry analysis, thereby revealing thedeuterium-labeled residues which correspond to the specific amino acidswith which the antibody interacts. See, e.g., Ehring (1999) AnalyticalBiochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem.73:256A-265A. Alternatively, in certain embodiments, the protein ofinterest binds to the antibody, followed by hydrogen-deuterium exchange.After dissociation of the antibody, the target protein is subjected toprotease cleavage and mass spectrometry analysis, thereby revealing thenon-deuterium-labeled residues which correspond to the specific aminoacids with which the antibody interacts. X-ray crystal structureanalysis can also be used to identify the amino acids within apolypeptide with which an antibody interacts.

The present invention further includes anti-CD28 and anti-EGFRantibodies that bind to the same epitope as any of the specificexemplary antibodies described herein (e.g. antibodies comprising any ofthe amino acid sequences as set forth in Tables 1, 3, 6, 9A, 9B and 9C).Likewise, the present invention also includes anti-CD28 and/or anti-EGFRantibodies that compete for binding to CD28 and/or EGFR with any of thespecific exemplary antibodies described herein (e.g. antibodiescomprising any of the amino acid sequences as set forth in Tables 1, 3,6, 9A, 9B and 9C herein).

The present invention also includes bispecific antigen-binding moleculescomprising a first antigen-binding domain that specifically binds humanCD28, and a second antigen binding fragment that specifically bindshuman EGFR, wherein the first antigen-binding domain binds to the sameepitope on CD28 as any of the specific exemplary CD28-specificantigen-binding domains described herein, and/or wherein the secondantigen-binding domain binds to the same epitope on EGFR as any of thespecific exemplary EGFR-specific antigen-binding domains describedherein.

In certain embodiments, the present invention includes bispecificantigen-binding molecules that interact with one or more of amino acidresidues 133-154 of EGFR as set forth in SEQ ID NO: 72, or amino acidresidues 345-368 of EGFR as set forth in SEQ ID NO: 70, or amino acidresidues 399-416 of EGFR as set forth in SEQ ID NO: 71.

Likewise, the present invention also includes bispecific antigen-bindingmolecules comprising a first antigen-binding domain that specificallybinds human CD28, and a second antigen binding fragment thatspecifically binds human EGFR, wherein the first antigen-binding domaincompetes for binding to CD28 with any of the specific exemplaryCD28-specific antigen binding domains described herein, and/or whereinthe second antigen-binding domain competes for binding to EGFR with anyof the specific exemplary EGFR-specific antigen-binding domainsdescribed herein.

One can easily determine whether a particular antigen-binding molecule(e.g., antibody) or antigen-binding domain thereof binds to the sameepitope as, or competes for binding with, a reference antigen-bindingmolecule of the present invention by using routine methods known in theart. For example, to determine if a test antibody binds to the sameepitope on CD28 (or EGFR) as a reference bispecific antigen-bindingmolecule of the present invention, the reference bispecific molecule isfirst allowed to bind to a CD28 protein (or EGFR protein). Next, theability of a test antibody to bind to the CD28 (or EGFR) molecule isassessed. If the test antibody is able to bind to CD28 (or EGFR)following saturation binding with the reference bispecificantigen-binding molecule, it can be concluded that the test antibodydoes not compete for binding to CD28 (or EGFR) with the referencebispecific antigen-binding molecule and/or that there is stericinterference between antibodies that are binding different sites on theantigen. On the other hand, if the test antibody is not able to bind tothe CD28 (or EGFR) molecule following saturation binding with thereference bispecific antigen-binding molecule, then the test antibodycompetes for binding to CD28 (or EGFR) with the reference bispecificantigen-binding molecule of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference bispecific antigen-binding molecule or if steric blocking (oranother phenomenon) is responsible for the lack of observed binding.Experiments of this sort can be performed using ELISA, RIA, Biacore,flow cytometry or any other quantitative or qualitative antibody-bindingassay available in the art. In accordance with certain embodiments ofthe present invention, two antigen-binding proteins compete for bindingto an antigen if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of oneantigen-binding protein inhibits binding of the other by at least 50%but preferably 75%, 90% or even 99% as measured in a competitive bindingassay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antigen-binding proteins may bind to the same epitopeif essentially all amino acid mutations in the antigen that reduce oreliminate binding of one antigen-binding protein reduce or eliminatebinding of the other. Two antigen-binding proteins may have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antigen-binding protein reduce or eliminatebinding of the other.

To determine if an antibody or antigen-binding domain thereof competesfor binding with a reference antigen-binding molecule, theabove-described binding methodology is performed in two orientations: Ina first orientation, the reference antigen-binding molecule is allowedto bind to a CD28 protein (or EGFR protein) under saturating conditionsfollowed by assessment of binding of the test antibody to the CD28 (orEGFR) molecule. In a second orientation, the test antibody is allowed tobind to a CD28 (or EGFR) molecule under saturating conditions followedby assessment of binding of the reference antigen-binding molecule tothe CD28 (or EGFR) molecule. If, in both orientations, only the first(saturating) antigen-binding molecule is capable of binding to the CD28(or EGFR) molecule, then it is concluded that the test antibody and thereference antigen-binding molecule compete for binding to CD28 (orEGFR). As will be appreciated by a person of ordinary skill in the art,an antibody that competes for binding with a reference antigen-bindingmolecule may not necessarily bind to the same epitope as the referenceantibody, but may sterically block binding of the reference antibody bybinding an overlapping or adjacent epitope.

Preparation of Antigen-Binding Domains and Construction of BispecificMolecules

Antigen-binding domains specific for particular antigens can be preparedby any antibody generating technology known in the art. Once obtained,two different antigen-binding domains, specific for two differentantigens (e.g., CD28 and EGFR), can be appropriately arranged relativeto one another to produce a bispecific antigen-binding molecule of thepresent invention using routine methods. (A discussion of exemplarybispecific antibody formats that can be used to construct the bispecificantigen-binding molecules of the present invention is provided elsewhereherein). In certain embodiments, one or more of the individualcomponents (e.g., heavy and light chains) of the multispecificantigen-binding molecules of the invention are derived from chimeric,humanized or fully human antibodies. Methods for making such antibodiesare well known in the art. For example, one or more of the heavy and/orlight chains of the bispecific antigen-binding molecules of the presentinvention can be prepared using VELOCIMMUNE™ technology. UsingVELOCIMMUNE™ technology (or any other human antibody generatingtechnology), high affinity chimeric antibodies to a particular antigen(e.g., CD28 or EGFR) are initially isolated having a human variableregion and a mouse constant region. The antibodies are characterized andselected for desirable characteristics, including affinity, selectivity,epitope, etc. The mouse constant regions are replaced with a desiredhuman constant region to generate fully human heavy and/or light chainsthat can be incorporated into the bispecific antigen-binding moleculesof the present invention.

Genetically engineered animals may be used to make human bispecificantigen binding molecules. For example, a genetically modified mouse canbe used which is incapable of rearranging and expressing an endogenousmouse immunoglobulin light chain variable sequence, wherein the mouseexpresses only one or two human light chain variable domains encoded byhuman immunoglobulin sequences operably linked to the mouse kappaconstant gene at the endogenous mouse kappa locus. Such geneticallymodified mice can be used to produce fully human bispecificantigen-binding molecules comprising two different heavy chains thatassociate with an identical light chain that comprises a variable domainderived from one of two different human light chain variable region genesegments. (See, e.g., US 2011/0195454 for a detailed discussion of suchengineered mice and the use thereof to produce bispecificantigen-binding molecules).

Bioequivalents

The present invention encompasses antigen-binding molecules having aminoacid sequences that vary from those of the described antibodies but thatretain the ability to bind CD28 and EGFR. Such variant moleculescomprise one or more additions, deletions, or substitutions of aminoacids when compared to parent sequence, but exhibit biological activitythat is essentially equivalent to that of the described antigen-bindingmolecules. Likewise, the antigen binding molecules-encoding DNAsequences of the present invention encompass sequences that comprise oneor more additions, deletions, or substitutions of nucleotides whencompared to the disclosed sequence, but that encode an antigen bindingmolecule that is essentially bioequivalent to the describedantigen-binding molecules of the invention. Examples of such variantamino acid and DNA sequences are discussed above.

The present invention includes antigen-binding molecules that arebioequivalent to any of the exemplary antigen-binding molecules setforth herein. Two antigen-binding proteins or antibodies are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of the exemplary bispecific antigen-bindingmolecules set forth herein may be constructed by, for example, makingvarious substitutions of residues or sequences or deleting terminal orinternal residues or sequences not needed for biological activity. Forexample, cysteine residues not essential for biological activity can bedeleted or replaced with other amino acids to prevent formation ofunnecessary or incorrect intramolecular disulfide bridges uponrenaturation. In other contexts, bioequivalent antibodies may includethe exemplary bispecific antigen-binding molecules set forth hereincomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention, according to certain embodiments, providesantigen-binding molecules that bind to human CD28 but not to CD28 fromother species. The present invention also provides antigen-bindingmolecules that bind to human EGFR but not to EGFR from other species.The present invention also includes antigen-binding molecules that bindto human CD28 and to CD28 from one or more non-human species; and/orantigen-binding molecules that bind to human EGFR and to EGFR from oneor more non-human species.

According to certain exemplary embodiments of the invention,antigen-binding molecules are provide which bind to human CD28 and/orhuman EGFR and may bind or not bind, as the case may be, to one or moreof mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat,sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzeeCD28 and or EGFR. For example, in a particular exemplary embodiment ofthe present invention, bispecific antigen-binding molecules are providedcomprising a first antigen-binding domain that binds human CD28 andcynomolgus CD28, and a second antigen-binding domain that specificallybinds human EGFR.

Immunoconjugates

The invention encompasses EGFRxCD28 antigen-binding proteins, e.g.,antibodies or antigen-binding fragments, such as REGN7075, REGN6321,REGN6322, REGN6323, or any combination of anti-EGFR HCVR pairing with anHCVR from any of the CD28 antibodies described herein, conjugated toanother moiety, e.g., a therapeutic moiety (an “immunoconjugate”). In anembodiment of the invention, an anti-EGFR or EGFRxCD28 antigen-bindingprotein, e.g., antibody or antigen-binding fragment, is conjugated toany of the further therapeutic agents set forth herein. As used herein,the term “immunoconjugate” refers to an antigen-binding protein, e.g.,an antibody or antigen-binding fragment, which is chemically orbiologically linked to another antigen-binding protein, a drug, aradioactive agent, a reporter moiety, an enzyme, a peptide, a protein ora therapeutic agent.

In certain embodiments, the therapeutic moiety may be a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Administration and Treatment

The present invention provides methods for administering an EGFRxCD28multispecific antigen-binding protein of the present invention, e.g.,REGN7075; REGN6321; REGN6322; REGN6323; or any combination of anti-EGFRHCVR pairing with an HCVR from any of the CD28 antibodies describedherein, or a pharmaceutical composition thereof, to a subject (e.g., ahuman for example, who suffers from a hyperproliferative disorder),comprising introducing the antigen-binding protein or pharmaceuticalcomposition into the body of the subject (e.g., a human), for example,parenterally. For example, the method comprises piercing the body of thesubject with a needle of a syringe and injecting the antigen-bindingprotein or pharmaceutical composition into the body of the subject,e.g., into the vein, artery, tumor, muscular tissue or subcutis of thesubject.

The mode of administration of an EGFRxCD28 or anti-EGFR antigen-bindingprotein or pharmaceutical composition thereof can vary. Routes ofadministration include parenteral, non-parenteral, oral, rectal,transmucosal, intestinal, parenteral; intramuscular, subcutaneous,intradermal, intramedullary, intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, intraocular, inhalation,insufflation, topical, cutaneous, intraocular, intravitreal, transdermalor intra-arterial.

The present invention also provides a vessel (e.g., a plastic or glassvial or ampule, e.g., with a cap or a chromatography column, hollow boreneedle or a syringe cylinder) comprising an EGFRxCD28 or anti-EGFRantigen-binding protein of the present invention or a pharmaceuticalcomposition thereof.

The present invention also provides an injection device comprising oneor more antigen-binding proteins (e.g., antibody or antigen-bindingfragment) that bind specifically to EGFR or EGFR and CD28 (EGFRxCD28) ora pharmaceutical formulation thereof. The injection device may bepackaged into a kit. An injection device is a device that introduces asubstance into the body of a subject via a parenteral route, e.g.,intramuscular, subcutaneous or intravenous. For example, an injectiondevice may be a syringe or an auto-injector (e.g., pre-filled with thepharmaceutical formulation) which, for example, includes a cylinder orbarrel for holding fluid to be injected (e.g., comprising the antibodyor fragment or a pharmaceutical formulation thereof), a needle forpiecing skin, blood vessels or other tissue for injection of the fluid;and a plunger for pushing the fluid out of the cylinder and through theneedle bore and into the body of the subject.

A pre-filled syringe is a syringe which has been filled with acomposition (e.g. a pharmaceutical composition comprising amultispecific antigen-binding protein and a pharmaceutically acceptablecarrier) prior to sale or transfer to an end-user, e.g., a physician orcare-giver, who is to administer the composition to a patient/subject.

Therapeutic Uses of the Antigen-Binding Molecules

The present invention provides methods for treating or preventing ahyperproliferative disease in a subject, comprising administering atherapeutically effective dose of EGFRxCD28 antigen-binding protein(e.g., REGN7075; REGN6321; REGN6322; REGN6323; or any combination ofanti-EGFR HCVR pairing with an HCVR from any of the CD28 antibodiesdescribed herein to the subject; optionally in association with a PD-1and/or PD-L1 inhibitor such as an antibody (e.g., pembrolizumab,nivolumab and/or cemiplimab) or antigen-binding fragment thereof. In anembodiment of the invention, the method includes the step of determiningwhether the cancer in the subject expresses EGFR. If such expression isobserved, then the EGFRxCD28 and/or anti-EGFR antigen-binding protein isadministered. For example, in an embodiment of the invention, the methodcomprises taking a biopsy of the cancer (e.g., which is performed by atreating physician) and either determining whether the cells of thecancer express EGFR or directing another individual or entity (e.g., onbehalf of the patient or subject) to perform such a determination and,if EGFR expression is present, then administering the anti-EGFR orEGFRxCD28 antigen-binding protein to the subject. In an embodiment ofthe invention, the physician directs some other individual or entity,for example, a pathologist (e.g., on behalf of the patient or subject)to perform the biopsy. In an embodiment of the invention, EGFRexpression is tested immunohistochemically (IHC) or by ELISA (enzymelinked immunosorbent assay).

The present invention includes methods including administering to asubject in need thereof a therapeutic composition comprising abispecific antigen binding molecule that specifically binds EGFR, orCD28 and EGFR. The therapeutic composition can comprise any of theantibodies or bispecific antigen-binding molecules as disclosed hereinand a pharmaceutically acceptable carrier or diluent.

The antibodies and bispecific antigen-binding molecules of the invention(and therapeutic compositions comprising the same) are useful, interalia, for treating any disease or disorder in which stimulation,activation and/or targeting of an immune response would be beneficial.In particular, the anti-EGFR, or EGFRxCD28 bispecific antigen-bindingmolecules of the present invention may be used for the treatment,prevention and/or amelioration of any disease or disorder associatedwith or mediated by EGFR expression or activity or the proliferation ofEGFR+ cells. The mechanisms of action by which the therapeutic methodsof the invention are achieved include killing of the cells expressingEGFR in the presence of effector cells, for example, T-cells. Cellsexpressing EGFR which can be inhibited or killed using the bispecificantigen-binding molecules of the invention include, for example, lungcancer cells.

As used herein, the term “subject” refers to a mammal (e.g., rat, mouse,cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, forexample, in need of prevention and/or treatment of an EGFR-expressingcancer. The subject may have an EGFR-expressing cancer, be predisposedto developing such a condition, and/or would benefit from an inhibitionor reduction in EGFR activity or a depletion of EGFR+ cells. In oneembodiment, the subject may have, or be at risk of developing, ahyperproliferative disease.

A hyperproliferative disease, for the purposes herein, refers to adisease characterized by abnormal, excessive and/or uncontrolled cellgrowth, e.g., wherein the cells express EGFR. For example,hyperproliferative diseases include EGFR-expressing cancers. A widerange of cancers express EGFR. Exemplary EGFR—expressing cancersinclude, but are not limited to esophageal carcinoma, lung squamous cellcarcinoma, lung adenocarcinoma, cervical squamous cell carcinoma,glioma, thyroid cancer, lung cancer (e.g., non-small cell lung cancer),colorectal cancer, colon cancer, bladder cancer, rectal cancer, head andneck cancer, stomach cancer, liver cancer, pancreatic cancer, renalcancer, urothelial cancer, prostate cancer, testis cancer, breastcancer, cervical cancer, endometrial cancer, ovarian cancer,gastroesophageal cancer, (e.g., gastroesophageal adenocarcinoma), andmelanoma. Accordingly, the antibodies and the bispecific antigen-bindingmolecules of the present invention can be used in treating a wide rangeof cancers.

Cancer characterized by solid tumor cells or cancerous blood cells,which may be an EGFR-expressing cancer e.g., wherein EGFR expression inthe cells of the particular subject to be treated has been confirmed,includes esophageal carcinoma, lung squamous cell carcinoma, lungadenocarcinoma, cervical squamous cell carcinoma, endometrialadenocarcinoma, bladder urothelial carcinoma, lung cancer (e.g.,non-small cell lung cancer), colorectal cancer, rectal cancer,endometrial cancer, skin cancer (e.g., head & neck squamous cellcarcinoma), brain cancer (e.g., glioblastoma multiforme), breast cancer,gastroesophageal cancer, (e.g., gastroesophageal adenocarcinoma),prostate cancer and/or ovarian cancer.

The antigen-binding molecules of the present invention may also be usedto treat, e.g., primary and/or metastatic tumors arising in the colon,lung, breast, renal cancer, and bladder (or from any cancer discussedherein). According to certain exemplary embodiments, the bispecificantigen binding molecules of the present invention are used to treat anovarian cancer.

The present invention also includes methods for treating residual cancerin a subject. As used herein, the term “residual cancer” means theexistence or persistence of one or more cancerous cells in a subjectfollowing treatment with an anti-cancer therapy.

According to certain aspects, the present invention provides methods fortreating a hyperproliferative disease, e.g., which is associated withEGFR expression (e.g., lung cancer), comprising administering one ormore of the anti-EGFR, or EGFRxCD28 bispecific antigen-bindingmolecules, as described herein to a subject, for example, after thesubject has been shown to be non-responsive to other types ofanti-cancer therapies. For example, the present invention includesmethods for treating a hyperproliferative disease such as lung cancercomprising administering an anti-EGFR or EGFRxCD28 bispecificantigen-binding molecule to a patient or subject 1 day, 2 days, 3 days,4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4months, 6 months, 8 months, 1 year, or more after the subject hasreceived the standard of care for patients suffering from cancer, e.g.,lung cancer. In other aspects, an anti-EGFR or EGFRxCD28 bispecificantigen-binding molecule of the invention comprising an IgG4 Fc domainis initially administered to a subject at one or more time points (e.g.,to provide robust initial depletion of ovarian cancer cells), followedby administration of an equivalent bispecific antigen-binding moleculecomprising a different IgG domain, such as an IgG1 Fc domain, atsubsequent time points. It is envisioned that the anti-EGFR or EGFRxCD28antibodies of the invention may be used in conjunction with otherbispecific antigen binding molecules, such as with an anti-EGFR/anti-CD3bispecific antibody. It is also envisioned that the bispecificantibodies of the invention will be used in conjunction with furthertherapeutic agents, for example, those that target PD-1, and othertargets. It may be advantageous to combine two bispecific antibodiesthat target the same tumor antigen (e.g., EGFR), but with one of thebispecifics targeting the CD3 on T-cells and the other bispecifictargeting a co-stimulator molecule like CD28. This combination may beused alone to enhance tumor cell killing, or may be used in combinationwith a checkpoint inhibitor.

An “effective” or “therapeutically effective” dose of EGFRxCD28 oranti-EGFR antigen-binding protein, e.g., antibody or antigen-bindingfragment, for treating or preventing a hyperproliferative disease, suchas an EGFR-expressing cancer, is the amount of the antigen-bindingprotein sufficient to alleviate one or more signs and/or symptoms of thedisease in the treated subject, whether by inducing the regression orelimination of such signs and/or symptoms or by inhibiting theprogression of such signs and/or symptoms. In an embodiment of theinvention, a therapeutically effective dose of anti-EGFR or EGFRxCD28antigen-binding protein is 0.1-2000 mg, e.g., 0.1 mg IV (intravenously)Q2W (every two weeks) to 2000 mg IV Q2W. The dose amount may varydepending upon the age and the size of a subject to be administered,target disease, conditions, route of administration, and the like. Incertain embodiments, the initial dose may be followed by administrationof a second or a plurality of subsequent doses of antigen-bindingprotein in an amount that can be approximately the same or less or morethan that of the initial dose, wherein the subsequent doses areseparated by 2 weeks.

The dose of antigen-binding molecule administered to a patient may varydepending upon the age and the size of the patient, target disease,conditions, route of administration, and the like. The preferred dose istypically calculated according to body weight or body surface area.Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted. Effective dosages andschedules for administering a bispecific antigen-binding molecule may bedetermined empirically; for example, patient progress can be monitoredby periodic assessment, and the dose adjusted accordingly. Moreover,interspecies scaling of dosages can be performed using well-knownmethods in the art (e.g., Mordenti et aL., 1991, Pharmaceut. Res.8:1351).

According to certain embodiments of the present invention, multipledoses of an antigen-binding molecule (e.g., an anti-CD28 antibody or abispecific antigen-binding molecule that specifically binds EGFR andCD28) may be administered to a subject over a defined time course. Themethods according to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an antigen-binding moleculeof the invention. As used herein, “sequentially administering” meansthat each dose of an antigen-binding molecule is administered to thesubject at a different point in time, e.g., on different days separatedby a predetermined interval (e.g., hours, days, weeks or months). Thepresent invention includes methods which comprise sequentiallyadministering to the patient a single initial dose of an antigen-bindingmolecule, followed by one or more secondary doses of the antigen-bindingmolecule, and optionally followed by one or more tertiary doses of theantigen-binding molecule.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the antigen-bindingmolecule of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of theantigen-binding molecule, but may differ from one another in terms offrequency of administration. In certain embodiments, however, the amountof an antigen-binding molecule contained in the initial, secondaryand/or tertiary doses varies from one another (e.g., adjusted up or downas appropriate) during the course of treatment. In certain embodiments,two or more doses are administered at the beginning of the treatmentregimen as “loading doses” followed by subsequent doses that areadministered on a less frequent basis (e.g., “maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered from one to several weeks after theimmediately preceding dose. The phrase “the immediately preceding dose,”as used herein, means, in a sequence of multiple administrations, thedose of antigen-binding molecule which is administered to a patientprior to the administration of the very next dose in the sequence withno intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antigen-binding molecule (e.g., an anti-CD28 antibody or abispecific antigen-binding molecule that specifically binds EGFR andCD28). For example, in certain embodiments, only a single secondary doseis administered to the patient. In other embodiments, two or moresecondary doses are administered to the patient. Likewise, in certainembodiments, only a single tertiary dose is administered to the patient.In other embodiments, two or more tertiary doses are administered to thepatient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. Alternatively, the frequency at which the secondaryand/or tertiary doses are administered to a patient can vary over thecourse of the treatment regimen. The frequency of administration mayalso be adjusted during the course of treatment by a physician dependingon the needs of the individual patient following clinical examination.

Diagnostic Uses

The bispecific antibodies of the present invention may also be used todetect and/or measure CD28 or EGFR, or CD28-expressing orEGFR-expressing cells in a sample, e.g., for diagnostic purposes. Forexample, anti-EGFR or EGFRxCD28 antibody or antigen-binding fragmentthereof, may be used to diagnose a condition or disease characterized byaberrant expression (e.g., over-expression, under-expression, lack ofexpression, etc.) of CD28 or EGFR. Exemplary diagnostic assays for CD28or EGFR may comprise, e.g., contacting a sample, obtained from apatient, with an antibody of the invention, wherein the antibody islabeled with a detectable label or reporter molecule. Alternatively, anunlabeled antibody can be used in diagnostic applications in combinationwith a secondary antibody which is itself detectably labeled. Thedetectable label or reporter molecule can be a radioisotope, such as ³H,¹⁴C, ³²p, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescent moiety such asfluorescein isothiocyanate, or rhodamine; or an enzyme such as alkalinephosphatase, betagalactosidase, horseradish peroxidase, or luciferase.Specific exemplary assays that can be used to detect or measure CD28 orEGFR in a sample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).Samples that can be used in CD28 or EGFR diagnostic assays according tothe present invention include any tissue or fluid sample obtainable froma patient which contains detectable quantities of CD28 or EGFR protein,or fragments thereof, under normal or pathological conditions.Generally, levels of CD28 or EGFR in a particular sample obtained from ahealthy patient (e.g., a patient not afflicted with a disease orcondition associated with abnormal CD28 or EGFR levels or activity) willbe measured to initially establish a baseline, or standard, level ofCD28 or EGFR. This baseline level of CD28 or EGFR can then be comparedagainst the levels of CD28 or EGFR measured in samples obtained fromindividuals suspected of having a CD28 or EGFR related disease orcondition.

Combinations and Pharmaceutical Formulations

The present invention provides compositions that include EGFRxCD28and/or anti-EGFR antigen-binding proteins and one or more ingredients;as well as methods of use thereof and methods of making suchcompositions. Pharmaceutical formulations (e.g., aqueous pharmaceuticalformulations that include water) comprising an EGFRxCD28 or anti-EGFRantigen-binding protein or the present invention and a pharmaceuticallyacceptable carrier or excipient are part of the present invention.

The present invention provides pharmaceutical compositions comprisingthe antigen binding molecules of the present invention. Thepharmaceutical compositions of the invention can be formulated withsuitable carriers, excipients, and other agents that provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, PA. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad,CA), DNA conjugates, anhydrous absorption pastes, oil-in-water andwater-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

To prepare pharmaceutical formulations of the EGFRxCD28 antigen-bindingproteins, e.g., antibodies and antigen-binding fragments thereof (e.g.,REGN7075; REGN6321; REGN6322; REGN6323; or any combination of anti-EGFRHCVR pairing with an HCVR from any of the CD28 antibodies describedherein, the antigen-binding protein is admixed with a pharmaceuticallyacceptable carrier or excipient. See, e.g., Remington's PharmaceuticalSciences and U.S. Pharmacopeia: National Formulary, Mack PublishingCompany, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman'sThe Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y. In an embodiment of the invention, the pharmaceuticalformulation is sterile. Such compositions are part of the presentinvention.

Pharmaceutical formulations of the present invention include anEGFRxCD28 or anti-EGFR antigen-binding protein and a pharmaceuticallyacceptable carrier including, for example, water, buffering agents,preservatives and/or detergents.

The scope of the present invention includes desiccated, e.g.,freeze-dried compositions, comprising an EGFRxCD28 or anti-EGFRantigen-binding protein, e.g., antibody or antigen-binding fragmentthereof, or a pharmaceutical formulation thereof that includes apharmaceutically acceptable carrier but substantially lacks water.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

As discussed herein, the present invention provides a vessel (e.g., aplastic or glass vial) or injection device (e.g., syringe, pre-filledsyringe or autoinjector) comprising any of the EGFRxCD28 or anti-EGFRantigen-binding proteins herein, e.g., antibodies or antigen-bindingfragments thereof, or a pharmaceutical formulation comprising apharmaceutically acceptable carrier or excipient thereof.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable and disposable pens and autoinjector delivery deviceshave applications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. See e.g., AUTOPEN™ (Owen Mumford,Inc., Woodstock, UK) or the HUMIRA™ Pen (Abbott Labs, Abbott Park, IL)

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Florida. In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline and otherisotonic solutions which may be used in combination with an appropriatesolubilizing agent. Injectable oily mediums are also part of the presentinvention. Such oily mediums may be combined with a solubilizing agent.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 0.1 to about 2000 mg per dosage form in aunit dose; especially in the form of injection.

The present invention includes compositions and therapeutic formulationscomprising any of the exemplary antibodies and bispecificantigen-binding molecules described herein in combination with one ormore additional therapeutically active components, and methods oftreatment comprising administering such combinations to subjects in needthereof.

Exemplary additional therapeutic agents that may be combined with oradministered in association with an antigen-binding molecule of thepresent invention include, e.g., chemotherapy (e.g., anti-cancerchemotherapy, for example, paclitaxel, docetaxel, vincristine,cisplatin, carboplatin or oxaliplatin), radiation therapy, checkpointinhibitors that target PD-1 (e.g., an anti-PD-1 antibody such aspembrolizumab, nivolumab, or cemiplimab (see U.S. Pat. No. 9,987,500)),CTLA-4, LAG3, TIM3, and others, costimulatory agonist bivalentantibodies that target molecules such as GITR, OX40, 4-1 BB, andothers), other costimulatory CD28 bispecific antibodies, andmultispecific (e.g., bispecific) antibodies and antigen-bindingfragments thereof that bind a tumor associated antigen (e.g., MUC16(mucin 16), PSMA, or STEAP2) and, for example, CD3 (MUC16xCD3, PSMAxCD3,or STEAP2xCD3). Exemplary bispecific antibodies comprising an antigenbinding domain that binds CD3 include, but are not limited to thosedescribed in, e.g., WO2017/053856A1, WO2014/047231A1, WO2018/067331A1and WO2018/058001A1. EGFR is expressed in a wide range of cancers.Accordingly, the bispecific anti-EGFRxCD28 antibodies of the presentinvention can be used in combination with a wide range of bispecificantibodies comprising an antigen-binding domain that binds CD3 intreatments of various cancers.

In a further embodiment of the invention, a further therapeutic agentthat is administered to a subject in association with an EGFRxCD28and/or anti-EGFR antigen-binding protein, e.g., antibody orantigen-binding fragment thereof, is administered to the subject inaccordance with the Physicians' Desk Reference 2003 (Thomson Healthcare;57^(th) edition (Nov. 1, 2002)) or the approved prescribing informationnormally provided with the particular agent.

Methods for treating or preventing an EGFR expressing cancer in asubject in need of said treatment or prevention by administering atherapeutically effective dose amount EGFRxCD28 and/or anti-EGFRantigen-binding protein, in association with a further therapeutic agentare part of the present invention.

The term “in association with” indicates that components, an EGFRxCD28or anti-EGFR antigen-binding protein, e.g., antibody or antigen-bindingfragment thereof of the present invention, along with a furthertherapeutic agent, such as cemiplimab, can be formulated into a singlecomposition, e.g., for simultaneous delivery, or formulated separatelyinto two or more compositions (e.g., a kit including each component).Components administered in association with each another can beadministered to a subject at a different time than when the othercomponent is administered; for example, each administration may be givennon-simultaneously (e.g., separately or sequentially) at intervals overa given period of time as part of a treatment regimen. Separatecomponents administered in association with each another may also beadministered sequentially, though essentially simultaneously, during thesame administration session. Moreover, the separate componentsadministered in association with each another may be administered to asubject by the same or by a different route.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention.

Example 1: Construction of Anti-EGFRxCD28 Antibodies Generation ofAnti-EGFR Antibodies

Anti-EGFR antibodies were obtained by directly administering anEGFR-expressing cell line (A431) with an adjuvant to stimulate theimmune response, to a VELOCIMMUNE® mouse comprising DNA encoding humanimmunoglobulin heavy and a universal light chain variable regions. Thatis, the antibodies produced in this mouse have different heavy chainvariable regions but essentially identical light chain variable domains.

The antibody immune response was monitored by an EGFR-specificimmunoassay. When a desired immune response was achieved, Anti-EGFRantibodies were isolated directly from antigen-positive B cells withoutfusion to myeloma cells, as described in U.S. Pat. No. 7,582,298.

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-EGFR antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers for Parental EGFR MonoclonalAntibodies (mAb) Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3LCVR LCDR1 LCDR2 LCDR3 mAb12999P2 2 4 6 8 16 18 20 22 mAb13008P2 30 3234 36 16 18 20 22 mAb35193P2 40 42 44 46 16 18 20 22 mAb13006P2 50 52 5456 16 18 20 22

TABLE 2 Nucleic Acid Sequence Identifiers for Parental EGFR MonoclonalAntibodies (mAb) Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3LCVR LCDR1 LCDR2 LCDR3 mAb12999P2 1 3 5 7 15 17 19 21 mAb13008P2 29 3133 35 15 17 19 21 mAb35193P2 39 41 43 45 15 17 19 21 mAb13006P2 49 51 5355 15 17 19 21

Generation of Anti-CD28 Antibodies

Anti-CD28 antibodies were obtained by immunizing a VELOCIMMUNE® mouse(i.e., an engineered mouse comprising DNA encoding human Immunoglobulinheavy and universal light chain variable regions) with human CD28protein fused to the Fc portion of mouse IgG2a, or with cells expressingCD28 or with DNA encoding CD28.

The antibody immune response was monitored by a CD28-specificimmunoassay. When a desired immune response was achieved, anti-CD28antibodies were isolated directly from antigen-positive B cells, asdescribed in U.S. Pat. No. 7,582,298.

Table 3 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-CD28 antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 4.

Certain biological properties of the exemplary anti-CD28 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

TABLE 3 Amino Acid Sequence Identifiers for Parental CD28 MonoclonalAntibodies (mAb) Antibody SEQ ID NOs: Designation HCVR HCDR1 HCDR2 HCDR3LCVR LCDR1 LCDR2 LCDR3 mAb14226 10 12 6 14 16 18 20 22 mAb14193 59 60 6162 16 18 20 22 mAb14216 63 64 65 66 16 18 20 22

TABLE 4 Nucleic Acid Sequence Identifiers for Parental CD28 AntibodiesAntibody SEQ ID NOS: Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2LCDR3 mAb14226 9 11 5 13 15 17 19* 21 mAb14193 15 17 19 21 mAb14216 1517 19 21 *ggggcaagt (SEQ ID NO: 19)Generation of Bispecific Antibodies (bsAbs) that Bind 0028 and EGFR

Bispecific antibodies comprising an anti-EGFR-specific binding domainand an anti-0028-specific binding domain were constructed using standardmethodologies, wherein the anti-EGFR antigen binding domain and theanti-AD28 antigen binding domain each comprise different, distinct HCVRspaired with a common LCVR. In some instances the bispecific antibodieswere constructed utilizing a heavy chain from an anti-CD28 antibody, aheavy chain from an anti-EGFR antibody and a common light chaincomprising the components, amino acid and nucleic acid sequencesencoding the antibodies as shown below in Tables 5, 6, 7 and 8.Additional bispecific antibodies that bind to EGFR and 0028 may beprepared using the parental monoclonal antibodies having thedesignations shown in Tables 9A, 9B3 and 90.

TABLE 5 Summary of Antibody Designations for HCVR Arms of Anti-EGFR xAnti-CD28 Bispecific Antibodies Anti-EGFR Anti-CD28 Antigen-BindingAntigen-Binding Domain Domain Bispecific Antibody (Parental Antibody(Parental Antibody Designation Designation) Designation) bsAb7075 (Alsoreferred to as mAb12999P2 mAb14226 REGN7075 or H4sH24623D) bsAb6321(Also referred to as mAb13008P2 mAb14226 REGN6321) bsAb6322 (Alsoreferred to as mAb35193P2 mAb14226 REGN6322) bsAb6323 (Also referred toas mAb13006P2 mAb14226 REGN6323)

TABLE 6 Amino Acid Sequences of Anti-EGFR × Anti-CD28 BispecificAntibodies Bispecific Anti-EGFR Anti-CD28 Common Light AntibodyAntigen-Binding Domain Antigen-Binding Domain Chain Variable RegionDesignation HCVR HCDR1 HCDR2 HCDR3 HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1LCDR2 LCDR3 bsAb7075 2 4 6 8 10 12 6 14 16 18 20 22 bsAb6321 30 32 34 3610 12 6 14 16 18 20 22 bsAb6322 40 42 44 46 10 12 6 14 16 18 20 22bsAb6323 50 52 54 56 10 12 6 14 16 18 20 22

TABLE 7 Nucleic Acid Sequences Encoding Anti-EGFR × Anti-CD28 BispecificAntibodies Anti-EGFR Anti-CD28 Common Bispecific Antigen-BindingAntigen-Binding Light Chain Variable Designation Domain Domain RegionAntibody HCVR HCDR1 HCDR2 HCDR3 HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2LCDR3 bsAb7075 1 3 5 7 9 11 5 13 15 17 19 21 bsAb6321 29 31 33 35 9 11 513 15 17 19 21 bsAb6322 39 41 43 45 9 11 5 13 15 17 19 21 bsAb6323 49 5153 55 9 11 5 13 15 17 19 21

TABLE 8 Amino acid and nucleotide sequences for full lengthimmunoglobulin chains of bispecific antibodies bsAb7075, bsAb6321,bsAb6322 and bsAb6323 Bispecific Antibody HC (EGFR) HC (CD28) LC (EGFR &CD28) Designation D P D P D P bsAb7075 23 24 25 26 27 28 bsAb6321 37 3825 26 27 28 bsAb6322 47 48 25 26 27 28 bsAb6323 57 58 25 26 27 28 D =Nucleotide sequence of DNA encoding indicated sequence P = amino acid ofpolypeptide for the indicated sequence Numbers refer to SEQ ID NOs forthe indicated sequence HC is the full length heavy chain for theindicated antibody LC is the full length light chain for the indicatedantibody

Additional bispecific antibodies comprising one HCVR from a parentalEGFR antibody and the other HCVR arm from a parental CD28 antibody maybe made using the techniques described herein. The parental EGFRantibodies used to generate these additional anti-EGFR X anti-CD28bispecific antibodies have HCVR sequences described in WO2014/004427.The CD28 parental antibodies used to generate these additional anti-EGFRX anti-CD28 bispecific antibodies have the amino acid sequencesdescribed above in Table 3. These anti-EGFR and anti-CD28 bindingdomains (pairings) are shown below in Tables 9A, 9B3 and 90.

TABLE 9A Summary of Parental Antibody Designations for HCVR Arms ofAnti-EGFR x Anti-CD28 Additional Bispecific Antibodies Anti-EGFRAnti-CD28 Antigen-Binding Domain Antigen-Binding Domain (ParentalAntibody (Parental Antibody Designation and Designation and Sequencesfound in Sequences found WO2014/004427) herein in Table 3) mAb085NmAb14226 mAb086N mAb14226 mAb136P mAb14226 mAb141P mAb14226 mAb142PmAb14226 mAb143P mAb14226 mAb144P mAb14226 mAb145P mAb14226 mAb147PmAb14226 mAb151P mAb14226 mAb153P mAb14226 mAb155P mAb14226 mAb157PmAb14226 mAb158P mAb14226 mAb159P mAb14226 mAb161P mAb14226 mAb163PmAb14226 mAb169P mAb14226 mAb171P mAb14226

TABLE 9B Summary of Parental Antibody Designations for HCVR Arms ofAnti-EGFR x Anti-CD28 Additional Bispecific Antibodies Anti-CD28Anti-EGFR Antigen- Antigen-Binding Binding Domain Domain (ParentalAntibody (Parental Antibody Designation and Designation and Sequencesfound in Sequences found WO2014/004427) herein in Table 3) mAb085NmAb14193 mAb086N mAb14193 mAb136P mAb14193 mAb141P mAb14193 mAb142PmAb14193 mAb143P mAb14193 mAb144P mAb14193 mAb145P mAb14193 mAb147PmAb14193 mAb151P mAb14193 mAb153P mAb14193 mAb155P mAb14193 mAb157PmAb14193 mAb158P mAb14193 mAb159P mAb14193 mAb161P mAb14193 mAb163PmAb14193 mAb169P mAb14193 mAb171P mAb14226

TABLE 9C Summary of Parental Antibody Designations for HCVR Arms ofAnti-EGFR x Anti-CD28 Additional Bispecific Antibodies Anti-EGFRAnti-CD28 Antigen-Binding Domain Antigen-Binding Domain (ParentalAntibody (Parental Antibody Designation and Designation and Sequencesfound in Sequences found WO2014/004427) herein in Table 3) mAb085NmAb14216 mAb086N mAb14216 mAb136P mAb14216 mAb141P mAb14216 mAb142PmAb14216 mAb143P mAb14216 mAb144P mAb14216 mAb145P mAb14216 mAb147PmAb14216 mAb151P mAb14216 mAb153P mAb14216 mAb155P mAb14216 mAb157PmAb14216 mAb158P mAb14216 mAb159P mAb14216 mAb161P mAb14216 mAb163PmAb14216 mAb169P mAb14216 mAb171P mAb14216

The bispecific antibodies described in the following examples consist ofbinding arms known to bind to human soluble heterodimeric hCD28 protein;and human EGFR (see Biacore binding data below). Exemplified bispecificantibodies were manufactured having a modified (chimeric) IgG4 Fc domainas set forth in US Patent Application Publication No. US20140243504A1,published on Aug. 28, 2014.

The bispecific antibodies created in accordance with the present Examplecomprise two separate antigen-binding domains (i.e., binding arms). Thefirst antigen-binding domain comprises a heavy chain variable regionderived from an anti-CD28 antibody (“CD28-VH”), and the secondantigen-binding domain comprises a heavy chain variable region derivedfrom an anti-EGFR antibody (“EGFR-VH”). Both the anti-EGFR and theanti-CD28 share a common light chain. The CD28-VH/EGFR-VH pairingcreates antigen-binding domains that specifically recognize CD28 on Tcells and EGFR on tumor cells.

Characterization of Bispecific Antibodies that Bind CD28 and EGFR

SPR Kinetics Method: Kinetics binding parameters were determined viasurface plasmon resonance (SPR) using a T-200 instrument and CM5 sensors(GE Healthcare). Double-referenced sensorgrams were globally fit to a1:1 binding model using Scrubber (BioLogics Software). The dissociationrate constant (kd) was determined by fitting the change in the bindingresponse during the dissociation phase, and the association rateconstant (ka) was determined by fitting analyte binding at differentconcentrations. The K_(D) was calculated from the ratio of the kd andka. The dissociative half-life (t½) was calculated as In2/kd andconverted to minutes.

EGFR Specifics Method: An anti-hFc monoclonal Ab (REGN2567) was coupledto a CM5 sensor surface using traditional EDC/NHS coupling chemistry.200-250 RUs of EGFRxCD28 Ab were captured on this surface. A six-point,three-fold dilution series of hEGFR.mmH starting at 30 nM was injectedover this surface at a flow rate of 50 uL/min for 5 minutes. Surface wasregenerated with a 10 second pulse of 20 mM H3PO4. Dissociation wasmeasured for 10 minutes. Sensorgrams were processed and fit as describedabove.

CD28 Capture Method Specifics: An anti-mFc polyclonal Ab (GE) wascoupled to a CM5 sensor surface using traditional EDC/NHS couplingchemistry. ˜30RUs of hCD28.mFc were captured on this surface. A sixpoint, three-fold dilution series of EGFRxCD28 Ab starting at 90 nM wasinjected over this surface at a flow rate of 50 uL/min for 4 minutes.Dissociation was measured for 5 minutes. Surface was regenerated with a40 second injection of 10 mM glycine, pH 1.5. Sensorgrams were processedand fit as described above.

Biacore analysis showed that EGFRxCD28 bound hEGFR with a K_(D) of˜9.3E-10 and hCD28 with a K_(D) of ˜5.2E-08 (Tables 10 and 11).

TABLE 10 Biacore Kinetics of Binding to EGFR mAB 30 nM Capture hEGFR.mmht½ AbPID (RU) Bind (RU) Ka (1/Ms) Kd (1/s) K_(D) (M) (min) Biacore 37ºC. Binding to Human EGFR-mmh REGN7075 245.6 ± 0.7 72.67 4.41E+054.12E−04 9.33E−10 4.24 Biacore 25º C. Binding to Human EGFR-mmh REGN7075209.6 ± 0.8 55.45 1.54E+05 4.33E−04 2.80E−09 26.69

TABLE 11 Biacore Kinetics of Binding to CD28 hCD28. mFc Capture 90 nMmAb t½ AbPID (RU) Bind (RU) Ka (1/Ms) Kd (1/s) K_(D) (M) (min) Biacore37º C. Binding to Human CD28-mFc REGN7075 39.8 ± 0.2 25.0 2.54E+051.31E−02 5.17E−08 0.9 Biacore 25º C. Binding to Human CD28-mFc REGN707532.6 ± 0.2 27.1 2.94E+05 3.14E−03 1.07E−08 3.7

Next, using co-cultures of human T-cells containing peripheral bloodmononuclear cells (PBMCs) and PEO-1, the ability of EGFRxCD28 to inducecellular cytotoxicity and T-cell activation was tested. Human peripheralblood mononuclear cells (PBMCs) were isolated from a healthy donorleukocyte pack. PBMC isolation was accomplished by density gradientcentrifugation using 50 mL SepMate™ tubes following the manufacturer'srecommended protocol. Briefly, 15 mL of FicollPaque PLUS was layeredinto 50 mL SepMate tubes, followed by addition of 30 mL of leukocytesdiluted 1:2 with D-PBS. Subsequent steps were followed according toSepMate manufacturer's protocol. CD3+ T-cells were subsequently isolatedfrom PBMC's using an EasySep Human T-Cell Isolation Kit from StemCellTechnologies and following manufacturer's recommended instructions.Isolated CD3⁺ T-cells were frozen in FBS containing 10% DMSO at aconcentration of 50×10⁶ cells per vial.

EGFRxCD28 significantly increased the ability of MUC16xCD3 (data notshown) to induce T-cell killing of tumor cells, from 18.8% to 71.5%(FIG. 1C). Consistent with this enhancement of T-cell cytotoxicity,EGFRxCD28 also boosted the T-cell activation as assessed byCD25-expression and IFNγ cytokine release (FIGS. 1D and 1E). Notably,EGFRxCD28 in the presence of non-targeted CD3 binding control (absenceof “signal 1”) had no effect on T-cell cytotoxicity or activation (FIGS.1C-1E).

Example 2: Over-Expression of a Natural CD28 Ligand on Tumor CellsSynergizes with PD-1 mAb Treatment to Induce CD8 T Cell-DependentDurable Anti-Tumor Immunity In Vivo

To determine if CD28 engagement by its natural ligand(s) couldpotentiate the anti-tumor efficacy of PD-1 mAb in vivo, MC38 tumor cellswere engineered to over-express CD86, one of the co-stimulatory ligandsfor CD28. Specifically, to generate tumor cell lines engineered toexpress co-stimulatory ligands, the pLVX lentiviral plasmid with EF1apromoter encoding mouse CD86 or empty vector and a puromycin resistancegene (pLVX.EF1a.CD86-puro and pLVX.EF1a.EV-puro, respectively) was usedto transfect HEK293T cells, facilitating the production of viralparticles, which were subsequently used to infect MC38 (National CancerInstitute, Laboratory of Tumor Immunology & Biology). Engineered celllines expressing CD86 were isolated by fluorescence-activated cellsorting (FACS). Cells were maintained under conditions recommended byATCC in the presence of 0.5 μg/ml Puromycin. Resulting cell lines weredesignated MC38/CD86 and MC38/EV.

Combination of MC38/CD86 cells and anti-PD-1 mAb treatment significantlyinhibited tumor growth (FIG. 2A), resulting in complete tumor regressionassociated with robust survival benefit (FIG. 2B) when compared with anegative control MC38 cells transfected with an empty vector control(MC38/EV). Depletion of CD8⁺ T-cells during the course of treatmentcompletely abrogated the anti-tumor efficacy elicited by combininganti-PD-1 mAb therapy with MC38/CD86 cells demonstrating a dependence onCD8⁺ T-cells (FIG. 2C). Of note, tumor free mice that were initiallyimplanted with MC38/CD86 cells and treated with anti-PD-1 mAb rejected asecond MC38 parental tumor that was implanted more than 60 days afterthe implantation of the primary tumor, indicating the presence of aT-cell memory response (FIG. 2D). Consequently, these data demonstratethat the synergistic effect of constitutive expression of a CD28 ligandand anti-PD-1 therapy can result in a durable CD8-dependent anti-tumorimmunity in vivo.

Example 3: Administration of EGFRxCD28 Antibodies in Combination withAnti-PD-1 Antibodies Synergistically Controls and Eradicates Mouse HumanTumor Xenografts, V_(H) Engrafted with Human CD34⁺ Stem Cells

The effectiveness of EGFRxCD28 bispecific antibody in combination withPD-1 blocking Ab (REGN2810 (cemiplimab), E. Burova et al.,Characterization of the Anti-PD-1 Antibody REGN2810 and Its AntitumorActivity in Human PD-1 Knock-In Mice. Mol Cancer Ther 16, 861-870(2017)) was tested using human tumor xenograft model. The engraftment ofhuman immune cell population was validated by FACS (FIG. 3A). A431epidermoid carcinoma tumor cells (obtained from ATCC) were implantedsubcutaneously in SIRPA^(h/h) TPO^(h/m) Rag2^(−/−)II2rg^(−/−)that wereengrafted with fetal liver CD34⁺ cells. Mice were segregated into 4groups based on fetal liver donor, human immune cell engraftmentfrequency and sex. Mice were dosed by intraperitoneal injection 2× perweek starting on the day of implant (day 0) with isotype control, 5mg/kg of EGFRxCD28, 10 mg/kg of PD-1 or combination. Antibodyinjection(s) were then administrated every 2-3 days through theexperiment. Tumors were measured two dimensionally (length×width) andtumor volume was calculated (length×width²×0.5). Mice were euthanizedwhen the tumor reached a designated tumor end-point (tumor volume>2000mm³ or tumor ulceration).

The expression of EGFR and PD-L1 on A431 tumor cells were validated byFACS (FIG. 3B). Each group was treated intra-peritoneally (IP) startingon the day of tumor challenge with:

-   -   (1) 10 mg/kg human Isotype Control No. 1+5 mg/kg human Isotype        Control No. 2    -   (2) 10 mg/kg anti-human PD-1 (REGN2810; cemiplimab)+5 mg/kg        human Isotype Control No. 2    -   (3) 5 mg/kg EGFRxCD28 (REGN7075)+10 mg/kg human Isotype Control        No. 1 or    -   (4) 5 mg/kg EGFRxCD28 (REGN7075)+10 mg/kg anti-human PD-1        (REGN2810). Antibody injection(s) were then administrated 2× per        week through the experiment. Tumors were measured two        dimensionally (length×width) and tumor volume was calculated        (length×width²×0.5). Mice were euthanized when the tumor reached        a designated tumor end-point (tumor volume>2000 mm³ or tumor        ulceration).        Isotype Control No. 1 and Isotype Control No. 2 are negative        control antibodies that bind Fel d1.

The size of the A431 tumor sizes over time in each treatment group areset forth in FIG. 4 . EGFRxCD28 in combination with anti-PD-1synergistically controlled and eradicated human tumor xenografts inhumanized mice. EGFRxCD28 monotherapy was able to delay tumor growthsignificantly and combination with anti-PD-1 Ab further inhibited thetumor progression. When a control PSMAxCD28 Ab was used in the sametreatment setting, tumor growth inhibition with or without anti-PD1treatment was not observed (FIG. 5 ). A lack of PSMA expression on A431tumor cells was validated by FACS (FIG. 6 ).

These data demonstrated that anti-PD1 monotherapy caused limitedanti-tumor response and anti-EGFR monotherapy caused partialanti-tumoral response whereas combination therapy exhibited a strong,robust anti-tumoral immune response.

Example 4: Administration of EGFRxCD28 Antibodies as MonotherapyControls and Eradicates Human A431 Tumor Xenografts in NSG MiceEngrafted with Human PBMC Cells

A431 epidermoid carcinoma tumor cells (obtained from ATCC) were mixedwith human PBMC and implanted subcutaneously in NSG mice (NOD/SCID/γ_(c)^(null)). Mice were treated with either a prophylactic (treatment on day0, 3, 7) or therapeutic (day 3, 7, 10) protocol.

For the prophylactic protocol, mice were segregated into 4 treatmentgroups. Each group was treated intra-peritoneally (IP) with:

-   -   (1) 5 mg/kg control Ab EGRvIIIxCD3 (bs17664D)    -   (2) 5 mg/kg EGFRxCD28 (REGN7075)    -   (3) 0.5 mg/kg EGFRxCD28 (REGN7075)    -   (4) 0.05 mg/kg EGFRxCD28 (REGN7075).

For therapeutic protocol, mice were segregated into 3 treatment group.Each group was treated with:

-   -   (1) 5 mg/kg control Ab EGRvIIIxCD3 (bs17664D)    -   (2) 5 mg/kg EGFRxCD28 (REGN7075)    -   (3) 0.05 mg/kg EGFRxCD28 (REGN7075).

Tumors were measured two dimensionally (length×width) and tumor volumewas calculated (length×width²×0.5). Mice were euthanized when the tumorreached a designated tumor end-point (tumor volume>2000 mm³ or tumorulceration).

The average tumor size in mice treated in each treatment group aresummarized in FIGS. 7A and 7B. These data demonstrated that in aprophylactic setting, a strong anti-tumor response is observed at allthree doses tested whereas the response was less than complete in atherapeutic setting.

Example 5: Administration of EGFRxCD28 Antibodies as MonotherapyControls and Eradicates Human A549 Tumor Xenografts in NSG MiceEngrafted with Human PBMC Cells

A549 lung adenocarcinoma tumor cells (obtained from ATCC) were implantedsubcutaneously in NSG mice (NOD/SCID/γ_(c) ^(null)) on day 0. Human PBMCwas engrafted on day 3 intra-peritoneally. Mice were segregated into 4treatment groups. Each group was treated intra-peritoneally (IP) with:

-   -   (1) 10 mg/kg control Ab EGRvlllxCD3 (bs17664D)    -   (2) 1 mg/kg EGFRxCD28 (REGN7075)    -   (3) 10 mg/kg EGFRxCD28 (REGN7075).

Ab treatment was administrated on day 15, 22 and 29. Tumors weremeasured two dimensionally (length×width) and tumor volume wascalculated (length×width²×0.5). Mice were euthanized when the tumorreached a designated tumor end-point (tumor volume>2000 mm³ or tumorulceration).

The average tumor sizes in mice treated in each treatment group aresummarized in FIG. 8 . A significant dose-dependent anti-tumor responsewas observed under a therapeutic setting with an established tumor.

Example 6: EGFRxCD28 Co-Stimulatory Bispecific Antibody Binds to CD28+and EGFR+ Cells

Flow cytometry analysis was utilized to determine binding of EGFRxCD28(REGN6323) to Jurkat cells (CD28⁺ cells) and to PEO1 cells (EGFR+cells). Binding of the antibody to each cell type was observed. SeeFIGS. 1A and 1B.

Cell lines endogenously expressing EGFR (PEO1, EGFR+) were labeled with1 μM of Violet Cell Tracker and plated overnight at 37° C. Separately,human PBMCs (New York Blood Center) or cynomolgus monkey PBMCs (Covance,Cranford NJ) were plated in supplemented RPMI media at 1×10⁶ cells/mLand incubated overnight at 37° C. in order to enrich for lymphocytes bydepleting adherent macrophages, dendritic cells, and some monocytes. Thenext day, the target cells were co-incubated with adherent cell-depletednaïve human PBMC (Effector/Target cell 4:1 ratio) and a serial dilutionof either TSAxCD3 or non-targeting CD3-based bispecific, alone or incombination with a fixed concentration (2.5 μg/ml) of a TSAxCD28bispecific for 96 hours at 37° C. Post incubation, the cells wereremoved from the cell culture plates using an enzyme-free celldissociation buffer and analyzed by Flow Cytometry (FACS).

For FACS analysis, cells were stained with a viability far red celltracker (Invitrogen) and directly conjugated antibodies to CD2, CD4, CD8and CD25 (BD). Samples were run with calibration beads for cellcounting. For the assessment of specificity of killing, target cellswere gated as Violet cell tracker positive populations. Percent of livetarget cells was calculated as follows: % viable cells=(R1/R2)*100,where R1=% live target cells in the presence of antibody, and R2=% livetarget cells in the absence of test antibody. T cell activation wasmeasured by the percent of activated (CD25⁺) T cells out of CD2⁺/CD4⁺ orCD2⁺/CD8⁺ T cells. T cell count was measured by calculating the numberof live CD4⁺ or CD8⁺ cells per calibration bead.

The levels of cytokines accumulated in the media were analyzed using theBD cytometric Bead Array (CBA) human Th1/Th2/Th17 Cytokine kit,following the manufacturer's protocol.

Example 7: Intra-Tumoral T-Cell Cluster Analysis

To examine whether treatment has significant impact on human T-cells'activation in the A431 tumor xenograft model, intra-tumoral T-cells wereprofiled. CITRUS (cluster identification, characterization, andregression), a method that independently stratifies statisticallysignificant different T cell clusters, was used to identify respondinghuman CD8+/CD4⁺ T-cell clusters upon single and combination treatment.

For flow cytometry analysis of in vivo experiments, tumors wereharvested, single cell suspensions were prepared, and red blood cellswere lysed using ACK Lysis buffer (ThermoFisher Scientific). Live/deadcell discrimination was performed using Live/dead fixable blue dead cellstaining kit (ThermoFisher Scientific). Samples were acquired onSymphony (BD Bioscience) and analyzed using Cytobank software (Cytobank,Santa Clara, CA). Analysis were performed with equal numbers of eventsper sample. The range in events was determined by the sample with thefewest events acquired. To cluster T cells automatically based onspecific markers, CITRUS analysis from Cytobank was used.

Both EGFRxCD28 (REGN7075) and anti-PD-1 Ab (cemiplimab) single agenttreatment reduced CD8⁺ T-cell cluster C1, which expresses a high levelof PD-L1 and ICOS (see FIGS. 9A and 9B). PD-1 blockade alone robustlydrove the expansion of a CD8⁺ T-cell cluster C2 with a highly activatedphenotype (High level of CD2, CD86, GITR, TIGIT and ICOS).Interestingly, more diverse CD4⁺ T cell clusters that responded tosingle or combination treatment (FIGS. 9C and 9D) were observed. Onlycombination treatment significantly expanded cluster C2 withproliferating effector memory like phenotype (High Ki67, low CD45RA andCCR7) and less exhausted effector memory like cluster C3 (Low CD38,CD45RA and CCR7). Similar to CD8⁺ T-cells, combination treatmentsignificantly reduced the cluster C1 with higher PDL1 expression level.PD-1 blockade monotherapy drove the expansion of effector memory cellswith higher CD38 expression level (C4), resembling a more activatedphenotype. EGFRxCD28 monotherapy maintained the small pool of naïve likeCD4⁺ T-cells in the tumor (C5), which may be further primed/activatedupon anti-PD1 Ab treatment.

These data showed that a human xenograft tumor model in humanized miceprovided a powerful platform to test EGFRxCD28 bi-specific antibody asmonotherapy and in combination with a PD-1 blockade reagent. Usinghigh-dimensional FACS analysis and a fully automated clusteringidentification method made it possible to further dissect theunderlining immune mechanism in human immune system reconstituted mice.Although PD1 blockade alone was able to drive potent expansion of CD8+and CD4⁺ T-cells with an activated phenotype, it was not sufficient topromote memory development and drive significant tumor inhibition. Whencombined with EGFRxCD28 treatment, a balanced T-cell activation statecan be achieved and drive potent anti-tumor response.

Example 8: EGFRxCD28 Alone or in Combination with Anti-PD1 Therapy doesnot Induce Systemic T-Cell Activation in Comparison to CD28 Superagonistin Cynomolgus Monkeys

To evaluate the tolerability of EGFRxCD28 bispecifics (REGN7075) alone,or the potential for synergistic pharmacology in combination withanti-PD1 antibody, studies in cynomolgus monkeys were conducted. Threemonkeys per treatment group received a single dose (10 mg/kg) ofEGFRxCD28 alone or in combination with REGN2810 (cemiplimab) (10 mg/kg)via intravenous infusion (combination groups received sequentialinfusions).

The cynomolgus monkey study was conducted in accordance with IACUCguidelines. For studies with EGFRxCD28, female cynomolgus monkeys(Macaca fascicularis) studies were carried out at SNBL USA (accreditedby the Association for Assessment and Accredation of Laboratory AnimalCare, AAALAC; Animal Welfare Assurance issued by Office of LaboratoryAnimal Welfare, OLAW; registered with the United States Department ofAgriculture, USDA and institutional animal care and use committee,IACUC). For flow cytometry, blood was collected from peripheral vein ofrestrained, conscious animals into potassium EDTA tubes. 100 μl of wholeblood was stained with the indicated antibodies. Flow cytometric dataacquisition was conducted using FACS Canto II. The absolute counts ofthe individual populations were calculated from their relativepercentages as derived from the parent/grandparent population gate andthe total parent/grandparent population counts from a validatedhematology analyzer according to the formula: Absolute population count(x10³/μL)=(population relative % x total parent/grandparent populationcount)/100. For serum cytokines, blood was collected into serumseparator tubes with anticoagulant. Serum separated via centrifugationin centrifuge set to 4° C. Analyzed using the MSD U-Plex platform (IL-2,4, 6, 8, 10, TNFα and IFNγ).

Assessment of toxicity was based on clinical observations, qualitativefood consumption, body weight, and vital signs (body temperature, heartrate, pulse oximetry, and respiration rate), and clinical and anatomicpathology upon completion of the experiment. Blood samples werecollected for cytokine and FACS immunophenotyping analysis. EGFRxCD28alone or in combination with cemiplimab were well tolerated and all theanimals survived to the time of scheduled necropsy. There was no testarticle related clinical-observations observed (data not shown). Nochanges in organ weights were found, nor were any macroscopic changesnoted at the terminal necropsy (data not shown). Furthermore, nosignificant cytokine release, T-cell marginalization or activation wereobserved (FIGS. 10A-10C). In contrast, significant cytokine release,lymphocyte marginalization and T-cell activation was seen in monkeysadministered a CD28 “superagonist” (data not shown). No significanttreatment-related histological changes were observed in animals thatwere administered EGFRxCD28 alone or in combination with cemiplimab.

Example 9: Dose Dependent Anti-Tumor Response Mediated by EGFR X CD28Bispecific Antibody (REGN7075) and Anti-PD-1 Antibody (REGN2810)Combination Therapy

Materials and Methods

NCI-H292

The NCI-H292 cell line is a human mucoepidermoid pulmonary carcinomacell line isolated from a 32-year old female patient (ATCC CRL-1848).The cell line was maintained in RPMI-1640 with 10% FBS supplemented withPSG (penicillin, streptomycin, and glutamine).

Peripheral Blood Mononuclear Cells (PBMC)

Human PBMC were obtained from ReachBio, Cat. #0500-401, Donor #0190205was used in this study.

Procedure Used for Tumor Study:

Female NSG mice (age ˜10 weeks old) were used in the experiments. Micewere implanted subcutaneously with 4×10⁶ tumor cells, which were mixedwith 2×10⁶ PBMC. Tumor growth was monitored by caliper twice a weekthroughout the duration of the study. The antibodies indicated in Table12 were administered as monotherapy or in combination by intraperitonealinjection at stated dosages on indicated days post tumor implantation.Experiment was ended when mice have ulcerated tumor or tumor volumeexceeding 1500 cm³ in accordance with IACUC standards. StatisticalAnalysis is shown in Table 13. The data is summarized in FIGS. 11 and 12.

TABLE 12 Study Design Ab 1 dose Ab 2 dose Mice per Group Ab 1 (mg/kg) Ab2 (mg/kg) group A Isotype Control 1 Isotype Control 0.1 8 No. 1 No. 2 BREGN2810 1 Isotype Control 0.1 8 No. 2 C Isotype Control 1 REGN7075 0.18 No. 1 D Isotype Control 1 REGN7075 0.02 8 No. 1 E REGN2810 1 REGN70750.1 9 F REGN2810 1 REGN7075 0.02 9 Isotype Control No.1 and IsotypeControl No. 2are negative control antibodies that bind Fel d1 REGN7075is the anti-EGFR X CD28 bispecific antibody also referred to as bsAb7075REGN2810 is an anti-PD-1 antibody

TABLE 13 Day 32 statistical analysis: Mixed-effects model (REML)analysis Tukey's multiple comparisons test Summary P value Isotype vsREGN2810 (1 mg/kg) + **** <0.0001 REGN7075 (0.1 mg/kg) Isotype vsREGN2810 (1 mg/kg) + ** 0.0073 REGN7075 (0.02 mg/kg) REGN2810 (1 mg/kg)vs REGN7075 * 0.0131 (0.1 mg/kg) REGN2810 (1 mg/kg) vs **** <0.0001REGN2810 (1 mg/kg) + REGN7075 (0.1 mg/kg) REGN2810 (1 mg/kg) vs ****<0.0001 REGN2810 (1 mg/kg) + REGN7075 (0.02 mg/kg) REGN7075 (0.1mg/kg) + **** <0.0001 REGN2810 (1 mg/kg) + REGN7075 (0.1 mg/kg) REGN7075(0.1 mg/kg) + * 0.0340 REGN2810 (1 mg/kg) + REGN7075 (0.02 mg/kg)REGN7075 (0.02 mg/kg) + **** <0.0001 REGN2810 (1 mg/kg) + REGN7075 (0.1mg/kg) REGN7075 (0.02 mg/kg) + **** <0.0001 REGN2810 (1 mg/kg) +REGN7075 (0.02 mg/kg)

Summary

The data, as shown in FIGS. 11 and 12 , demonstrated that when tested inthis model, the most robust anti-tumor response is observed whenREGN7075 (anti-EGFR X anti-CD28) is combined with an anti-PD-1 antibody(REGN2810), as compared to the results obtained when either antibody isused alone.

Example 10A: Flow Cytometry Binding Titration Using REGN6323

Flow cytometric analysis was utilized to determine binding of anti-EGFRX anti-CD28 bispecific antibodies to several cancer cell lines (A375,22RV1, PEO1, CAPAN2, SWi1990, H292), Human and Cynomolgus T cells andJurkat cells followed by detection with a labeled anti-human secondaryIgG antibody. Briefly, 1×10⁵ cells/well were incubated for 30 minutes at4° C. with a serial dilution of EGFRxCD28 bispecific antibodies orControl Antibody (a human IgG4 antibody that binds a human antigen withno cross-reactivity to human or cynomolgus CD28), ranging from 100 nM to1.5 μM for human and cynomolgus T cells, and ranging from 133 nM to 2 μMfor EGFR expressing cells. After incubation, the cells were washed twicewith cold PBS containing 1% filtered FBS and a Fluorophore-conjugatedanti-human secondary antibody was added to the cells and incubated foran additional 30 minutes. Live/dead dye was added to Human andCynomolgus T cells incubations. Wells containing no antibody orsecondary only were used as a control.

After incubation with EGFR expressing cells, cells were washed,re-suspended in 200 μL cold PBS containing 1% filtered FBS and analyzedby flow cytometry on a BD FACS Canto II.

After incubation with Human or Cynomolgus T cells, cells were washed,and stained with a cocktail of anti-CD2, ant-CD16, anti-CD4, andanti-CD8 antibodies in Brilliant Stain Buffer for an extra 20 minincubation at 4° C. After wash, cells were re-suspended in 200 μL coldPBS containing 1% filtered FBS, gated in a Live/CD2+/CD4+/CD16− orLive/CD2+/CD8+/CD16− gate and analyzed by Flow cytometry on a BD FACSLSR-Fortessa-X20.

Results of Flow Cytometry Binding Titration (See Tables 14A and 14B andFIGS. 13A, 13B and 13C)

The binding of an anti-EGFR X anti-CD28 bispecific antibody (REGN6323)to the surface of human T cells and cynomolgus T cells was tested asdescribed above by flow cytometry. The results showed that REGN6323bound to both human and cynomolgus CD4⁺ and CD8⁺ T cells, withoutreaching saturation within the range of concentrations selected (FIG.13A).

The binding of an anti-EGFR X anti-CD28 bispecific antibody (REGN6323)to the surface of other cell lines expressing EGFR was also tested byflow cytometry. The cell lines used were Jurkat cells (See FIG. 13C),which are an immortalized line of human T lymphocyte cells; A375 cells,which are an epithelial melanoma cell line; 22RV1 cells, which are anepithelial prostate carcinoma; PEO1 cells, which are an ovariancarcinoma cell line derived from ascites; CAPAN2, which is a pancreaticadenocarcinoma cell line; SW1990, which is a pancreatic adenocarcinomaderived from a metastatic site (spleen); and H292, which is a lungepithelial carcinoma cell line. The results show that REGN6323 bound toA375 cells with an EC50 of 1.48E-09M. REGN6323 bound to 22RV1 cells withan EC50 of 5.54E-10M. REGN6323 bound to PEO1 cells with an EC50 of6.67E-10. REGN6323 bound to CAPAN2 cells with an EC50 of 1.29E-09M.REGN6323 bound to SW1990 cells with an EC50 of 2.96E-09M and REGN6323bound to H292 cells with an EC50 of 6.74E-10 (See FIG. 13B).

The isotype control antibody did not exhibit any binding to human orcynomolgus T cells, nor did it bind to cell lines expressing EGFR.

These data suggest that REGN6323 is capable of binding a variety ofcancer cell lines expressing EGFR and as such, may be effective fortreating multiple cancer indications.

Example 10B: Flow Cytometry Binding Titration Using REGN6321 andREGN6322

An experiment was also done to determine whether other anti-EGFR Xanti-CD28 bispecific antibodies could also exhibit bind to multiple celltypes. Following the protocol described above, REGN6321 and REGN6322were tested in a similar manner. REGN6323 was also included in thisstudy.

The results, as shown in FIG. 13C, demonstrate that REGN6321, REGN6322and REGN6323 exhibit binding to a prostate cell line, 22RV1, as well asa cell line designated PEO1, which is an ovarian cell line. Theseresults demonstrated that these additional anti-EGFR X anti-CD28antibodies are capable of binding across other cancer cell linesexpressing EGFR.

TABLE 14 Summary of Flow Cytometry Binding Studies with Various EGFR XCD28 Bispecific Antibodies Anti-EGFR X Anti-CD28 Bispecific AntibodyBinding EC₅₀ [M] Designation CASKI A375 22RV1 PEO1 REGN6321 1.69E−085.42E−09 +, NC +, NC REGN6322 2.44E−08 4.64E−09 9.58E−09 5.77E−09REGN6323 2.35E−09 8.74E−10 1.19E−08 5.86E−09 REGN7075 2.05E−09 NT NT1.22E−09 NC: EC50 Not Calculated: NT: Antibody not tested

Example 11: Cytotoxicity as Measured by Flow Cytometry

In order to monitor the killing of cells expressing Tumor SpecificAntigens (TSA) of various sizes in the presence of a combination of ananti-TSA X anti-CD3 and an EGFRxCD28 antibody, 22RV1 (PSMA+, STEAP2+) orPEO1 (MUC16+) or CAPAN2 (MUC16+) or SW1990 (MUC16+) or H292 (MUC16+)cells were labeled with 1 uM of the fluorescent tracking dye Violet CellTracker. After labeling, cells were plated overnight at 37° C.Separately, human PBMCs were plated in supplemented RPMI media at 1×10⁶cells/mL and incubated overnight at 37° C. in order to enrich forlymphocytes by depleting adherent macrophages, dendritic cells, and somemonocytes. The next day, target cells were co-incubated with adherentcell-depleted naïve PBMC (Effector/Target cell 4:1 ratio), a serialdilution of a targeting TSAxCD3 bispecific antibodies or a controlTSAxCD3 bispecific antibody (concentration range: 66.7 nM to 0.2 μM) anda fixed concentration of an EGFRxCD28 costimulatory molecule REGN6323 at2.5 ug/ml (16.7 nM) for 96 hours at 37° C. Cells were removed from cellculture plates using Trypsin-EDTA dissociation buffer, and analyzed byFlow Cytometry on a FACS BD LSRFortessa-X20. For Flow Cytometryanalysis, cells were stained with a dead/live Near IR Reactive(Invitrogen) dye. 5E05 counting beads were added to each wellimmediately before Flow Cytometry analysis. 1E05 beads were collectedfor each sample. For the assessment of specificity of killing, cellswere gated on live Violet labeled populations. Percent of livepopulation was recorded and used for the calculation of survival.

T cell activation was assessed by incubating cells with directlyconjugated antibodies to CD2, CD4, CD8, CD25, and by reporting thepercent of late activated T cells out of total T cells (CD2+) or CD8⁺ Tcells.

Results of Flow Cytometry Based Cytotoxicity Assay (See Table 15 andFIGS. 14 and 15 )

The costimulatory EGFRxCD28 bispecific antibody REGN6323 was tested forits ability to enhance the cytotoxic potency of TSAxCD3 antibodiestargeting cell surface antigens of various sizes, and across severalcancer indications.

REGN6323 successfully enhanced the cytotoxic potency of various TSAxCD3bispecific antibodies targeting PSMA or MUC16 or STEAP2 in the presenceof unstimulated human PBMC. Target cell killing was enhanced in thepresence of the an EGFRxCD28+TSAxCD3 bispecific antibodies combinationcompared to TSAxCD3 single agent treatment, and cells were killed in adose-dependent manner with pM EC50s.

The observed target-cell lysis enhancement was associated withenhancement of CD25+ expression on T cells, again with pM EC50s.

TABLE 15 Kill T cell Kill T cell (TSAxCD3 activation (TSAxCD3 activationonly) (TSAxCD3 + (TSAxCD3 + %, only) %, EGFRxCD28) EGFRxCD28) EC₅₀ [M]EC₅₀ [M] %, EC₅₀ [M] %, EC₅₀ [M] 22RV1 (with 54%, 93%, 50%, 97%,STEAP2xCD3) 4.31E−11 3.86E−11 1.11E−11 6.27E−12 22RV1 (with 24%, N/C58%, 46%, 95%, PSMAxCD3) 7.28E−10 5.5E−11 2.53E−11 PEO1 (with 58%, 44%,79%, 60%, MUC16xCD3) 1.87E−10 1.51E−10 4.75E−11 1.71E−11 CAPAN2 (with Noactivity No activity 7%, 55%, MUC16xCD3) 5.29E−11 5.78E−11 SW1990 (with49%, 30%, 60%, 85%, MUC16xCD3) 3.14E−10 1.64E−10 2.31E−11 2.14E−11 H292(with No activity No activity 32%, 35%, MUC16xCD3) 3.96E−10 1.49E−10

Example 12: Epitope Mapping on EGFR with REGN7075, REGN6323 and REGN6322by HDX-MS

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) was performed todetermine the amino acid residues of the Epidermal Growth FactorReceptor (recombinant human EGFR, amino acid sequence in appendix)interacting with REGN7075 (mAb12999P2×mAb14226P2), mAb13006P (REGN6323EGFR arm parental), and mAb35193P (REGN6322 EGFR arm parental). Ageneral description of the HDX-MS method is set forth in e.g., Ehring(1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith(2001) Anal. Chem. 73:256A-265A. The results are shown in Tables 16, 17and 18.

The HDX-MS experiments were performed on an integrated HDX-MS platform,consisting of a Leaptec HDX PAL system for the deuterium labeling andquenching, a Waters Acquity M-Class (Auxiliary solvent manager) for thesample digestion and loading, a Waters Acquity M-Class (pBinary solventmanager) for the analytical gradient, and a Thermo Q Exactive HF massspectrometer for peptide mass measurement.

The labeling solution was prepared as PBS buffer in D₂O at pD 7.0 (10 mMphosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent to pH 7.4 at 25°C.). For deuterium labeling, 11 μL of 52.0 μM hEGFR.mmh (Regeneron inhouse protein REGN355) or hEGFR.mmh premixed with REGN7075 in 1:1 molarratio (Ag-Ab complex), 11 μL of 34.3 μM hEGFR.mmH (Regeneron in houseprotein REGN355) or hEGFR.mmH premixed with mAb13006P2 in 1:0.6 molarratio (Ag-Ab complex), 11 μL of 31.4 μM hEGFR.mmH (Regeneron in houseprotein REGN355) or hEGFR.mmH premixed with mAb35193P2 in 1:0.6 molarratio (Ag-Ab complex) were incubated at 20° C. with 44 μL of D₂Olabeling solution for various time-points in duplicates (e.g.,non-deuterated control=0 second; deuterium-labeled for 5 minutes and 10minutes). The deuteration reaction was quenched by adding 55 μL ofpre-chilled quench buffer (0.5 M TCEP-HCl, 8 M urea and 1% formic acid)to each sample for a 5-minute incubation at 20° C. The quenched sampleswere then injected into a Waters HDX Manager for online pepsin/proteaseXIII digestion. The digested peptides were separated by a C8 column (1.0mm×50 mm, NovaBioassays) at −9.5° C. with a 22-minute gradient from0%-90% B (mobile phase A: 0.5% formic acid and 4.5% acetonitrile inwater, mobile phase B: 0.5% formic acid in acetonitrile). The elutedpeptides were analyzed by a Thermo Q Exactive HF mass spectrometry inLC-MS/MS or LC-MS mode.

The LC-MS/MS data of undeuterated EGFR sample were searched against adatabase including hEGFR.mmH (REGN355) sequence and its reversedsequence using Byonic search engine (Protein Metrics). The searchparameters were set as default using non-specific enzymatic digestionand human glycosylation as common variable modification. The list ofidentified peptides was then imported into the HDX Workbench software(version 3.3) to calculate the deuterium uptake of each peptide detectedby LC-MS from all deuterated samples. For a given peptide, the centroidmass (intensity-weighted average mass) at each time point was used tocalculate the deuterium uptake (D) and percentage of deuterium uptake (%D).

DeuteriumUptake(D − uptake) = AverageMass(deuterated) − AverageMass(undeuterated)${{Percentage}{of}{deuterium}{uptake}\left( {\% D} \right)} = \frac{D - {uptake}{for}{peptide}{at}{each}{time}{point} \times 100\%}{{Maximum}D - {uptake}{of}{the}{peptide}}$

A total of 271 peptides from hEGFR.mmH (REGN355) were identified fromboth hEGFR.mmH alone and hEGFR.mmH in complex with REGN7075 samples,representing 84.4% sequence coverage of hEGFR.mmH. Any peptide whichexhibited a differential percent D-uptake value above 5% was defined assignificantly protected. Peptides corresponding to amino acids 345-368(LHILPVAFRGDSFTHTPPLDPQEL SEQ ID NO: 70) and 399-416 (LEIIRGRTKQHGQFSLAVSEQ ID NO: 71) on hEGFR.mmH were significantly protected by REGN7075(hEGFR.mmH residues are numbered according to hEGFR.mmH sequences).

A total of 341 peptides from hEGFR.mmH (REGN355) were identified fromboth hEGFR.mmH alone and hEGFR.mmH in complex with mAb13006P samples,representing 85.8% sequence coverage of hEGFR.mmH. Any peptide whichexhibited a differential percent D-uptake value above 5% was defined assignificantly protected. Peptides corresponding to amino acids 345-368(LHILPVAFRGDSFTHTPPLDPQEL SEQ ID NO: 70) and 399-416 (LEIIRGRTKQHGQFSLAVSEQ ID NO: 71) on hEGFR.mmH were significantly protected by mAb13006P(hEGFR.mmH residues are numbered according to hEGFR.mmH sequences in theappendix).

A total of 335 peptides from hEGFR.mmH (REGN355) were identified fromboth hEGFR.mmH alone and hEGFR.mmH in complex with mAb35193P samples,representing 85.5% sequence coverage of hEGFR.mmH. Any peptide whichexhibited a differential percent D-uptake value above 5% was defined assignificantly protected. Peptides corresponding to amino acids 133-154(CNVESIQWRDIVSSDFLSNMSM SEQ ID NO: 72) on hEGFR.mmH were significantlyprotected by mAb35193P (hEGFR.mmH residues are numbered according tohEGFR.mmH sequences).

TABLE 16 hEGFR.mmH peptides with significant protection upon formationof hEGFR.mmH-REGN7075 complex comparing to hEGFR.mmH alone 5 min 10 minhEGFR- hEGFR- hEGFR REGN7075 hEGFR REGN7075 hEGFR Residues Charge (+)Centroid MH⁺ Centroid MH⁺ ΔD Centroid MH⁺ Centroid MH⁺ ΔD Δ % D 345-3522 455.74 456.12 −0.38 455.78 456.19 −0.42 −19.3 346-351 1 650.00 650.39−0.38 650.06 650.50 −0.45 −16.9 346-352 1 797.35 797.97 −0.62 797.43798.14 −0.71 −20.3 346-352 2 399.07 399.28 −0.21 399.08 399.32 −0.24−14.1 346-355 2 563.57 564.01 −0.44 563.64 564.17 −0.53 −16.1 346-356 2607.27 607.74 −0.47 607.35 607.91 −0.56 −15.9 346-367 3 826.78 827.17−0.39 826.87 827.38 −0.51 −10.3 346-368 3 864.65 865.06 −0.41 864.81865.31 −0.50 −10.0 399-412 2 843.25 843.57 −0.32 843.38 843.77 −0.39−7.3 400-412 2 786.85 787.12 −0.27 786.97 787.33 −0.36 −7.0 400-412 3524.89 525.07 −0.18 524.96 525.19 −0.23 −6.9 400-416 2 972.10 972.37−0.26 972.15 972.62 −0.47 −6.0 401-412 2 722.21 722.47 −0.26 722.34722.71 −0.37 −7.7 401-412 3 481.82 482.02 −0.20 481.90 482.16 −0.26 −8.5

TABLE 17 hEGFR.mmH peptides with significant protection upon formationof hEGFR.mmH-mAb13006P complex comparing to hEGFR.mmH alone 5 min 10 minhEGFR- hEGFR- hEGFR mAb13006P hEGFR mAb13006P hEGFR Residues Charge (+)Centroid MH⁺ Centroid MH⁺ ΔD Centroid MH⁺ Centroid MH⁺ ΔD Δ % D 345-3522 455.74 456.16 −0.41 455.82 456.18 −0.36 −18.7 346-351 1 650.01 650.40−0.40 650.08 650.50 −0.42 −16.4 346-352 1 797.35 797.99 −0.64 797.47798.12 −0.65 −19.6 346-354 2 505.90 506.44 −0.54 505.99 506.57 −0.58−22.8 346-355 2 563.51 564.07 −0.56 563.62 564.18 −0.56 −19.6 346-356 2607.23 607.78 −0.55 607.34 607.92 −0.57 −17.3 346-367 3 826.80 827.22−0.42 826.93 827.37 −0.44 −10.0 346-368 2 1296.50 1297.08 −0.58 1296.771297.40 −0.63 −8.9 346-368 3 864.63 865.06 −0.43 864.80 865.29 −0.48−10.0 399-412 2 843.24 843.64 −0.41 843.32 843.76 −0.44 −8.7 400-412 2786.75 787.15 −0.40 786.84 787.29 −0.45 −9.6 400-412 3 524.83 525.10−0.28 524.88 525.18 −0.30 −9.6 400-415 2 922.54 922.99 −0.45 922.65923.09 −0.44 −7.9 400-416 2 972.04 972.46 −0.42 972.11 972.57 −0.46 −7.3401-412 2 722.13 722.56 −0.43 722.23 722.67 −0.44 −10.8 401-412 3 481.79482.09 −0.31 481.86 482.17 −0.32 −11.5

TABLE 18 hEGFR.mmH peptides with significant protection upon formationof hEGFR.mmH-mAb35193P complex comparing to hEGFR.mmH alone 5 min 10 minhEGFR- hEGFR- hEGFR mAb35193P hEGFR mAb35193P hEGFR Residues Charge (+)Centroid MH⁺ Centroid MH⁺ ΔD Centroid MH⁺ Centroid MH⁺ ΔD Δ % D 133-1391 793.31 793.75 −0.45 793.33 793.96 −0.64 −13.3 133-142 2 626.18 626.65−0.47 626.23 626.80 −0.57 −15.8 133-147 2 877.53 878.02 −0.49 877.56878.27 −0.71 −11.4 137-141 1 690.43 690.70 −0.28 690.48 690.86 −0.38−13.2 137-147 2 654.56 654.79 −0.22 654.63 654.91 −0.28 −6.9 138-147 2610.96 611.19 −0.23 611.06 611.31 −0.25 −7.4 140-142 1 476.68 476.72−0.03 476.70 476.81 −0.11 −8.4 143-148 1 668.39 668.72 −0.33 668.47668.88 −0.41 −11.3 148-152 1 612.31 612.48 −0.17 612.38 612.59 −0.21−7.5 148-154 2 1220.00 1220.45 −0.45 1220.17 1220.51 −0.34 −18.6

Amino acid sequence of hEGFR.mmh hEGFR(ECD).CPGG.MMH6 (REGN355): humanEGFR (amino acids L25-A647, Accession #NM_005228.4), with a C-terminalCPGG.myc epitope(E1-L10).GlyGly.myc epitope(E1-L10).SerGly.6XHis.SSG tag(mmh tag is underlined)

(SEQ ID NO: 69) LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIACPGGEQKLISEEDLGGEQKLISEEDLS GHHHHHHSSG

Example 13: Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of EGFRxCD28 Bispecific and Associated ParentalAntibodies to EGFR and CD28

EGFR Experimental Procedure

Equilibrium dissociation constants (K_(D) values) for hEGFR.mmH(REGN355) binding to captured anti-EGFRxCD28 bispecific Abs weredetermined using a real-time surface plasmon resonance biosensor using aBiacore 4000 instrument. The CM5 Biacore sensor surface was derivatizedby amine coupling with a monoclonal mouse anti-human Fc antibody(REGN2567, Lot #REGN2567-L1) to capture purified anti-EGFRxCD28bispecific Abs. This Biacore binding study was performed in a buffercomposed of 0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA, 0.05% v/vSurfactant P20 (HBS-EP running buffer). Different concentrations ofhEGFR.mmH with a C-terminal myc-myc-hexahistidine tag (REGN355) preparedin HBS-EP running buffer (ranging from 30 nM to 120 μM, 3-fold serialdilutions) were injected over the Ab captured surface at a flow rate of30 μL/minute. Association was monitored for 5 minutes, and thedissociation of hEGFR.mmH in HBS-EP running buffer was monitored for 10minutes. All of the binding kinetics experiments were performed at 25°C. Kinetic association (k_(a)) and dissociation (k_(d)) rate constantswere determined by fitting the real-time sensorgrams to a 1:1 bindingmodel using Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t½) werecalculated from the kinetic rate constants as:

K _(D)(M)=k _(d) /k _(a), and t½(min)=0.693/k _(d)/60

Binding kinetic parameters for hEGFR.mmH binding to purified EGFRxCD28Abs at 25° C. are shown below in Table 19.

CD28 Experimental Procedure

Equilibrium dissociation constants (K_(D) values) for purifiedbispecific Abs binding to captured hCD28.mFc (REGN2012) were determinedusing a real-time surface plasmon resonance biosensor using a BiacoreT-200 instrument. The CM5 Biacore sensor surface was derivatized byamine coupling with a polyclonal rabbit anti-moue Fc antibody (GEHealthcare, Cat #BR100838) to capture purified hCD28 with a C-terminalmouse Fc tag (hCD28.mFc, REGN2012). This Biacore binding study wasperformed in a buffer composed of 0.01 M HEPES pH 7.4, 0.15M NaCl, 3 mMEDTA, 0.05% v/v Surfactant P20 (HBS-EP running buffer). Differentconcentrations of bispecific Abs prepared in HBS-EP (ranging from 90 nMto 1.1 nM, 3-fold serial dilutions) were injected over the hCD28.mFccaptured surface at a flow rate of 50 μL/minute. Association wasmonitored for 4 minutes, and the dissociation of bispecific Abs inHBS-EP was monitored for 10 minutes. All of the binding kineticsexperiments were performed at 25° C. Kinetic association (k_(a)) anddissociation (k_(d)) rate constants were determined by fitting thereal-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curvefitting software. Binding dissociation equilibrium constants (K_(D)) anddissociative half-lives (t½) were calculated from the kinetic rateconstants as:

K _(D)(M)=k _(d) /k _(a), and t½(min)=0.693/k _(d)/60

Binding kinetic parameters for EGFRxCD28 Abs binding to hCD28.mFc at 25°C. are shown below in Table 20.

Tabulated Data Summary:

TABLE 19 hEGFR.mmH Binding Kinetics to anti-EGFR × CD28 bispecific mAbat 25° C. REGN #/ EGFR CD28 Common kd KD t½ Ab PID# Lot # Arm Arm Nameka (1/Ms) (1/s) (M) (min) REGN6321 REGN6321- 13008P5 14226P5 EGFR × CD287.30E+04 4.63E−05 6.34E−10 249.4 L4 REGN6322 REGN6322- 35193P2 14226P2EGFR × CD28 1.56E+05 1.46E−03 9.38E−09 7.9 L3 REGN6323 REGN6323- 13006P214226P2 EGFR × CD28 2.06E+05 7.74E−04 3.75E−09 14.9 L2

TABLE 20 hCD28.mFc Binding Kinetics to anti-EGFR × CD28 bispecific mAbat 25° C. REGN #/ EGFR CD28 Common kd KD t½ Ab PID # Lot # Arm Arm Nameka (1/Ms) (1/s) (M) (min) REGN6321 REGN6321- 13008P5 14226P5 EGFR × CD282.59E+05 2.81E−03 1.08E−08 4.1 L4 REGN6322 REGN6322- 35193P2 14226P2EGFR × CD28 2.61E+05 2.68E−03 1.03E−08 4.3 L3 REGN6323 REGN6323- 13006P214226P2 EGFR × CD28 2.65E+05 2.65E−03 1.00E−08 4.4 L2

Example 14: Characterization of EGFR x CD28 Bispecific Antibodies inLigand-Mediated Cell Growth Assay Using Engineered BaF3 CellsOverexpressing Human EGFR

The EGFR (ErbB1, HER1) receptor is a member of the receptor tyrosinekinase (RTK) family, which regulate cell proliferation, survival,differentiation and migration of multicellular organisms. The activationof the receptor occurs via binding of its soluble ligands, e.g. EGF orTGFβ, which drive the homo-dimerization and autophosphorylation of EGFR,leading eventually to the activation of a plethora of intracellularsignaling cascades such as Ras/MAPK, PLCγ1/PKC, PI3-kinase/Akt, and STATpathways (Wieduwilt et al. 2008; Cell Mol Life Sci. May; 65(10):1566-1584).

To study the blocking effects of anti-EGFR x CD28 antibodies on EGFRsignaling, a proliferation assay was deployed using an engineeredIL-3-dependent Ba/F3 murine hematopoietic cell line genetically modifiedto stably overexpress human epidermal growth factor receptor (EGFR—aminoacids M1 to A1210 of Genbank accession #NP_005219.2) (Kong et al. 2017;Oncotarget, Vol. 8, (No. 22), pp: 35488-35489). Engineered BaF3/hEGFRcells were stimulated with ligands (human EGF or TGF3) in the presenceof titrated antibodies and cell growth was measured by tritiumincorporation as a function of proliferating cells.

Experimental Procedures:

Engineered BaF3/hEGFR cells were grown in culture medium (RPM11640+10%FBS+Penicilin/Streptomycine/L-Glutamine+10 μg/mL mouse IL-3+0.5 μg/mLpuromycine), were starved in media (RPM11640+1% FBS) without IL-3, for24h before using them in cell-proliferation assays. Briefly, in 96-wellround-bottom tissue culture plates 12.5×10{circumflex over ( )}5cells/well were added to 1:3 serially diluted antibodies, ranging from15.2 μM to 100 nM including a no antibody containing control, in thepresence of constant ligands, hEGF (0.5 nM) or hTGFβ (0.5 nM). Plateswere incubated for 72h at 37° C./5% CO₂, and 0.3 μCi/well tritiatedthymidine was added to cells and plates were incubated for another 16hours. Thymidine, and therefore tritium, will be incorporated at higheramounts into newly synthesized DNA of the dividing cells. After theincubation, cells were harvested onto 96-well UniFilter plates and 30 μLof scintillation fluid was added to each well. Tritium incorporation wasmeasured as counts per minute (CPM) using the Microplate Scintillation &Luminescence Counter TopCount NXT instrument. Cells, antibodies andligands were prepared in assay media (Opti-MEM+0.1% Bovine SerumAlbumine) and all serial dilutions were tested in duplicate.

The IC₅₀/EC₅₀ values of the antibodies were determined from a4-parameter logistic equation over a 10-point dose-response curve usingGraphPad Prism™ software. Maximum inhibition of proliferation wasdetermined by the following equation:

${{Inhibition}{}(\%)} = {{100\%} - \frac{100\% \times \begin{pmatrix}{{{Mean}{CPM}{at}0.5{}{nM}{}{Ligand}} +} \\{{100{nM}{mAb}} - {{Background}{CPM}}}\end{pmatrix}}{\begin{matrix}\left( {{{Mean}{CPM}{at}05{nM}{Ligand}} +} \right. \\{{0{nM}{mAb}} - {{Background}{CPM}}}\end{matrix}}}$

Summary of Results:

EGFR x CD28 bispecifics (REGN6322; REGN6323 and REGN7075) and theircorresponding parental EGFR antibodies (mAb35193P2; mAb13006P2 andmAb12999P2) were tested alongside in-house generated comparator antibody(Erbitux) and isotype matched negative controls in the presence of 0.5nM hEGF or hTGFβ (see Table 21).

In the presence of 0.5 nM EGF:

None of the bispecific antibodies (REGN6322; REGN6323 and REGN7075)showed a dose-dependent inhibition of proliferation. Their maximalinhibition values ranged from 30.5 to −6.8% (negative values mean anincrease of proliferation). Whereas mAb35193P2, mAb12999P2 and in-houseErbitux inhibited the proliferation of BaF3/hEGFR cells with IC₅₀ andmaximal inhibition values of 2.55 nM/29.6%, 19.8 nM/91.8% and 6.69nM/97.6%, respectively. mAb13006P2 displayed maximal inhibition of93.2%, an IC₅₀ value could not be calculated. The negative isotypecontrol antibodies showed no inhibition as expected.

In the presence of 0.5 nM hTGFβ:

REGN6322 showed a 40.6% increase in proliferation with an EC₅₀ of 3.67nM, whereas REGN6323 and REGN7075 showed no response similar to thenegative isotype control antibodies. In contrast, mAb13006P2, mAb12999P2and in-house Erbitux inhibited the proliferation with IC₅₀ and maximalinhibition values of 6.55 nM/97.2%, 7.79 nM/95.9% and 1.38 nM/99.8%,respectively. mAb35193P2 displayed an increase of proliferation by 32.7%with an EC₅₀ value of 914 pM.

TABLE 21 Maximum Inhibition and Potency values of Antibodies In presenceof 0.5 nM hEGF In presence of 0.5 nM hTGFβ Inhibition InhibitionAntibodies [%] IC₅₀ [M] EC₅₀ [M] [%] IC₅₀ [M] EC₅₀ [M] REGN6322 30.5 ND−40.6 3.67E−09 REGN6323 28.5 ND −7.3 ND REGN7075 −6.8 ND 11.2 NDmAb35193P2 29.6 2.55E−09 −32.7 9.14E−10 mAb13006P2 93.2 NC 97.2 6.55E−09mAb12999P2 91.8 1.98E−08 95.9 7.79E−09 In-house 97.6 6.69E−09 99.81.38E−09 Erbitux Isotype 7.8 ND 18.9 ND Control I Isotype 14.1 ND −1.4ND Control II Abbreviations: ND: Not Determined; NC: Not Calculable; apotency value could not be determined by PRISM.

Example 15: Characterization of EGFR x CD28 Bispecific Antibodies inAllogeneic T-Cell Activation Assays Using NCI-H292 and Human PrimaryT-Cells

TABLE 22 Reagent/Antibody Information/Materials: AbPID/REGN# DescriptionREGN6322 EGFRxCD28 REGN6323 EGFRxCD28 REGN7075 EGFRxCD28 Non-TAA x CD28REGN6157 = non-TAA (tumor-associated antigen) x CD28 Cemiplimab REGN2810(PD-1 antibody) IsoC-1 Isotype control for Cemiplimab IsoC-2 Isotypecontrol CD28 bispecific

BACKGROUND

Two signals, “signal 1” & “signal 2,” are required for proper T cellactivation. “Signal 1” is induced by binding of the T cell receptor(TCR) on T cells to peptide-bound major histocompatibility complex (MHC)molecules on antigen presenting cells (APCs). Whereas, “signal 2” isprovided by engaging the co-stimulatory CD28 receptor on T cells withits ligands cluster of differentiation 80 or 86 (CD80/CD86) present onAPCs (Martin et al. 1986; June et al. 1987; Harding et al. 1992).Therefore, activation of CD28 signaling provides a targeted approach toenhance existing TCR signaling.

EGFRxCD28 bispecific antibodies are designed to mimick the naturalligands of CD28, by bridging EGFR⁺ target cells with CD28⁺ T cells, toprovide “signal 2” in order to enhance the activation of T cells inpresence of an existing “signal 1” in an allogeneic T cell activationassay. However, T cell activation can be inhibited by the ligation ofprogrammed cell death protein 1 receptor (PD-1) on T cells to its ligandPD-L1 on APCs. Ligated PD-1 leads to the recruitment of phosphatases toCD28 and the TCR complex (Zou et al. 2008, Francisco et al. 2010, Hui etal. 2017), which in turn counteract TCR signaling and CD28 stimulation.Thus, blockade of the PD-1/PD-L1 interaction with cemiplimab incombination with EGFRxCD28 bispecific antibodies may potentiate T cellfunction and promote killing of target cells such as in cancer.

Experimental Procedures:

The ability of EGFRxCD28 bispecific antibodies to activate human primaryT-cells by engaging EGFR and CD28 to deliver “signal 2”, as determinedby IL-2 release and T-cell proliferation, was evaluated in the presenceof the EGFR+/PD-L1⁺ human lung cancer cell line (NCI-H292) that providesan allogeneic TCR response sufficient to serve as “signal 1”.

Isolation of Human Primary CD3⁺ T Cells:

Human peripheral blood mononuclear cells (PBMCs) were isolated from ahealthy donor leukocyte pack from Precision for Medicine (Donor 555015)using the EasySep™ Direct Human PBMC Isolation Kit, following themanufacturers recommended protocol and frozen down. CD3⁺ T-cells wereisolated by thawing vials of frozen PBMCs. Donor PBMCs were enriched forCD3⁺ T-cells using an EasySep™ Human CD3⁺ T Cell Isolation Kit fromStemCell Technologies and following the manufacturer's recommendedinstructions.

IL-2 Release Assay:

Enriched CD3⁺ T-cells, resuspended in stimulation media, were added into96-well round bottom plates at a concentration of 1×10⁵ cells/well.Growth-arrested NCI-H292 cells, which endogenously express EGFR andPD-L1, were added to CD3⁺ T-cells at a final concentration of 5×10⁴cells/well. Subsequently, REGN6322, REGN6323, REGN7075, Non-TAAxCD28,and their matched isotype control (IsoC-2) were titrated from 0.76 μM to50 nM in a 1:4 dilution and added to wells. The final point of the10-point dilution contained no titrated antibody. Following addition oftitrated antibody, a constant 20 nM of either cemiplimab or its matchedisotype control (IsoC-1) was added to wells. Plates were incubated for96 hours at 37° C., 5% CO₂ and 50 □L total supernatant was removed and 5□L from collected supernatant was used for measuring IL-2. The amount ofIL-2 in assay supernatant was determined using AlphaLisa kit fromPerkinElmer following the manufacturer's protocol. The IL-2 measurementswere acquired on Perkin Elmer's multilabel plate reader Envision andvalues were reported as pg/mL. All serial dilutions were tested intriplicate.

The EC₅₀ values of the antibodies were determined from a four-parameterlogistic equation over a 10-point dose-response curve using GraphPadPrism™ software. Maximal IL-2 is given as the mean max response detectedwithin the tested dose range.

T-Cell Proliferation Assay:

After incubation of 96 hours at 37° C., 5% CO₂ and supernatant removal(see IL-2 release assay), 0.25 mCi/well tritiated thymidine was added tocells and plates were incubated for another 6-8 hours. Thymidine, andtherefore tritium, will be incorporated at higher amounts into newlysynthesized DNA of the dividing cells. After the incubation, cells wereharvested onto 96-well UniFilter plates and 30 μL of scintillation fluidwas added to each well. Tritium incorporation was measured as counts perminute (CPM) using the Microplate Scintillation & Luminescence CounterTopCount NXT instrument. All serial dilutions were tested in triplicate.

The EC₅₀ values of the antibodies were determined from a four-parameterlogistic equation over a 10-point dose-response curve using GraphPadPrism™ software. Maximal CPM is given as the mean max response detectedwithin the tested dose range.

Summary of Results (Table 23)

In the presence of cemiplimab EGFRxCD28 antibody treatment (REGN6322,REGN6323, and REGN7075) lead to higher IL-2 and proliferative responsecompared to Non-TAA x CD28 and IsoC-2, the respective matched isotypecontrol for CD28 bispecifics. Whereas, in the absence of cemiplimab,EGFRxCD28 antibody treatment leads to higher IL-2 and proliferativeresponse compared to Non-TAA x CD28 and IsoC-1, however the max valuesare lower.

TABLE 23 Maximum IL-2 release & Proliferation and Potency values ofAntibodies IL-2 Release Proliferation MAX EC₅₀ MAX EC₅₀ Antibodies(pg/mL) [M] (CPM) [M] REGN6322 + cemiplimab 21.8 NC 2382 NC REGN6323 +cemiplimab 36.7 NC 2345 3.13E−11 REGN7075 + cemiplimab 27.2 NC 32502.16E−11 Non-TAAxCD28 + cemiplimab 10.5 NC 1215 ND IsoC-2 + cemiplimab8.99 ND 1106 ND REGN6322 + IsoC-1 8.02 ND 1863 NC REGN6323 + IsoC-1 13.2ND 1665 2.29E−11 REGN7075 + IsoC-1 12.7 NC 1880 3.92E−11 Non-TAAxCD28 +IsoC-1 6.10 ND 632 ND IsoC-2 + IsoC-1 5.96 ND 875 ND Abbreviations: ND:Not Determined; NC: Not Calculable; a potency value could not bedetermined by PRISM.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1.-50. (canceled)
 51. A set of nucleic acid molecules encoding abispecific antigen-binding molecule, wherein the bispecificantigen-binding molecule comprises a first antigen-binding domain thatspecifically binds human CD28, and a second antigen-binding domain thatspecifically binds human EGFR, wherein the set of nucleic acid moleculescomprises: (a) a first nucleic acid molecule encoding a heavy chainvariable region (HCVR) comprising three heavy chain complementaritydetermining regions HCDR1, HCDR2, and HCDR3, comprising the amino acidsequences of SEQ ID NOs: 12, 6 and 14, respectively; (b) a secondnucleic acid molecule encoding a heavy chain variable region (HCVR)comprising three heavy chain complementarity determining regions HCDR1,HCDR2, and HCDR3, comprising the amino acid sequences of SEQ ID NOs: 4,6 and 8, respectively; and (c) a third nucleic acid molecule encoding alight chain variable region (LCVR) comprising three light chaincomplementarity determining regions LCDR1, LCDR2 and LCDR3, comprisingthe amino acid sequences of SEQ ID NOs: 18, 20, and 22, respectively.52. The set of nucleic acid molecules of claim 51, wherein: (a) thefirst nucleic acid molecule encodes a HCVR comprising the amino acidsequence of SEQ ID NO: 10; (b) the second nucleic acid molecule encodesa HCVR comprising the amino acid sequence of SEQ ID NO: 2; and (c) thethird nucleic acid molecule encodes a LCVR comprising the amino acidsequence of SEQ ID NO:
 16. 53. The set of nucleic acid molecules ofclaim 51, wherein the bispecific antigen-binding molecule is abispecific antibody, the first nucleic acid molecule encodes a firstheavy chain, the second nucleic acid molecule encodes a second heavychain, and the third nucleic acid molecule encodes a light chain. 54.The set of nucleic acid molecules of claim 53, wherein one of the heavychains comprises a C_(H)3 domain comprising a H435R (EU numbering)modification and/or a Y436F (EU numbering) modification.
 55. The set ofnucleic acid molecules of claim 53, wherein the first heavy chain, thesecond heavy chain, or both the first and second heavy chains comprise ahuman IgG1 heavy chain constant region.
 56. The set of nucleic acidmolecules of claim 53, wherein the first heavy chain, the second heavychain, or both the first and second heavy chains comprise a human IgG4heavy chain constant region.
 57. The set of nucleic acid molecules ofclaim 53, wherein the first heavy chain comprises the amino acidsequence of SEQ ID NO: 26, the second heavy chain comprises the aminoacid sequence of SEQ ID NO: 24, and the light chain comprises the aminoacid sequence of SEQ ID NO:
 28. 58. The set of nucleic acid molecules ofclaim 51, wherein the first nucleic acid molecule comprises thenucleotide sequences of SEQ ID NO: 11, 5, and 13; the second nucleicacid molecule comprises the nucleotide sequences of SEQ ID NO: 3, 5 and7; and the third nucleic acid molecule comprises the nucleotidesequences of SEQ ID NO: 17, 19 and
 21. 59. The set of nucleic acidmolecules of claim 52, wherein the first nucleic acid molecule comprisesthe nucleotide sequence of SEQ ID NO: 9, the second nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO: 1, and thethird nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO:
 15. 60. The set of nucleic acid molecules of claim 53, wherein thefirst nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO: 25, the second nucleic acid molecule comprises the nucleotidesequence of SEQ ID NO: 23, and the third nucleic acid molecule comprisesthe nucleotide sequence of SEQ ID NO:
 27. 61. An expression vector or aset of expression vectors comprising the set of nucleic acid moleculesof claim
 51. 62. An isolated host cell comprising the expression vectoror the set of expression vectors of claim
 61. 63. The isolated host cellof claim 62, wherein the isolated host cell is a Chinese hamster ovary(CHO) cell.
 64. An isolated host cell comprising the set of nucleic acidmolecules of claim
 51. 65. An isolated host cell comprising the set ofnucleic acid molecules of claim
 52. 66. A method of producing abispecific antigen-binding molecule that binds human CD28 and humanEGFR, comprising culturing the host cell of claim 62 under conditionspermitting production of the bispecific antigen-binding molecule, andrecovering the bispecific antigen-binding molecule so produced.
 67. Themethod of claim 66, further comprising formulating the bispecificantigen-binding molecule as a pharmaceutical composition with a suitablecarrier.